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Sample records for extracellular atp eatp

  1. The Danger Signal Extracellular ATP Is an Inducer of Fusobacterium nucleatum Biofilm Dispersal

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    Ding, Qinfeng; Tan, Kai Soo

    2016-01-01

    Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP) is an example of a danger associated molecular pattern (DAMP) employed by mammalian cells to elicit inflammatory and damage healing responses. Although, the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors such as eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also showed higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a host danger signaling molecule to detach

  2. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

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    Elizabeth eMedina-Castellanos

    2014-11-01

    Full Text Available The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation. During this response, reactive oxygen species (ROS are produced by the NADPH oxidase (Nox1/NoxR complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK pathways by eATP, Ca2+ and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP. Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals.

  3. Extracellular ATP Selectively Upregulates Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 and Ecto-5'-Nucleotidase by Rat Cortical Astrocytes In Vitro.

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    Brisevac, Dusica; Adzic, Marija; Laketa, Danijela; Parabucki, Ana; Milosevic, Milena; Lavrnja, Irena; Bjelobaba, Ivana; Sévigny, Jean; Kipp, Markus; Nedeljkovic, Nadezda

    2015-11-01

    Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis.

  4. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU).

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    Zhao, Ronglan; Liang, Dongchun; Sun, Deming

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases.

  5. Extracellular ATP-induced NO production and its dependence on membrane Ca2+ flux in Salvia miltiorrhiza hairy roots

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    Wu, Shu-Jing; Wu, Jian-Yong

    2008-01-01

    Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca2+ and H2O2 in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10–500 μM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10–100 μM but decreased from 100–200 μM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPγS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca2+ antagonists, but not affected by a protein kinase inhibitor. ATP also induced H2O2 production, which was dependent on both Ca2+ and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca2+ level, which was dependent on NO but not H2O2. The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca2+ and ROS signalling. PMID:18977749

  6. Extracellular ATP induces albuminuria in pregnant rats

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    Faas, M.M.; van der Schaaf, G.; Borghuis, T.; Jongman, R.M.; van Pampus, Maria; de Vos, P.; van Goor, Harry; Bakker, W.W.

    2010-01-01

    BACKGROUND: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy. METHODS: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula.

  7. Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena

    ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......Y receptors. Previously, our group has shown that cholinergic stimulation of acini caused ATP release into ducts and ATP is an important regulator of ductal functions by being involved in ion and fluid secretion. Pancreatic duct cells are exposed to a number of stimuli, well known to induce ATP...... release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological...

  8. Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena

    ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......Y receptors. Previously, our group has shown that cholinergic stimulation of acini caused ATP release into ducts and ATP is an important regulator of ductal functions by being involved in ion and fluid secretion. Pancreatic duct cells are exposed to a number of stimuli, well known to induce ATP...... release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological...

  9. Extracellular ATP and P2X7 receptors in neurodegeneration.

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    Le Feuvre, Rosalind; Brough, David; Rothwell, Nancy

    2002-07-05

    Neuronal injury and cell death in the central nervous system (CNS) are underlying features of neurodegenerative disorders. However, our understanding of the fundamental mechanisms involved is still limited. Inflammatory processes mediated by cytokines, and interleukin-1 (IL-1) in particular, play a significant role in neuronal death following pathological insults. Despite this growing area of research, very little is known about the factors regulating the expression, cleavage and release of interleukin-1 in the brain. Recent studies on immune cells demonstrate that extracellular ATP can act as a potent stimulus for the maturation and release of interleukin-1beta, via activation of P2X7 receptors. Stimulation of P2X7 receptors with ATP has dramatic cytotoxic properties and a wider role in neurodegenerative processes is possible. This review discusses the potential involvement of extracellular ATP and P2X7 receptors as regulators of interleukin-1-mediated neuropathologies and thus as a mediator of cell death following pathological insults.

  10. In vivo imaging demonstrates ATP release from murine keratinocytes and its involvement in cutaneous inflammation after tape stripping.

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    Takahashi, Toshiya; Kimura, Yutaka; Niwa, Kazuki; Ohmiya, Yoshihiro; Fujimura, Taku; Yamasaki, Kenshi; Aiba, Setsuya

    2013-10-01

    Adenosine 5'-triphosphate (ATP) release from keratinocytes has been observed in various stress models in vitro, but studies demonstrating epidermal ATP release in vivo are limited. To visualize extracellular ATP (eATP) in vivo, we developed enhanced green-emitting luciferase immobilized on agarose beads (Eluc-agarose). Subcutaneous injection of Eluc-agarose together with ATP into the dorsal skin of BALB/c mice following intraperitoneal luciferin injection produced detectable and measurable bioluminescence using an in vivo imaging system. Using Eluc-agarose, we demonstrated in vivo that bright bioluminescence was observed from 1 to 20 minutes after repeated tape stripping of murine skin. This bioluminescence was suppressed by the local administration of apyrase. Eluc-agarose bioluminescence was observed only in tape-stripped skin with transepidermal water loss (TEWL) between 100 and 140 g m(2) h(-1), indicating a loss of bioluminescence with excessive tape stripping (TEWL>140 g m(-2) h(-1)). Histologically, tape-stripped skin with detectable eATP had a viable epidermis and a subepidermal neutrophil infiltrate, and administration of apyrase reduced the inflammatory infiltrate. Neither a viable epidermis nor an upper dermal neutrophil infiltrate was observed after excessive tape stripping. These results suggest that tape stripping prompts ATP release from viable keratinocytes, which facilitates inflammatory cell migration. Eluc-agarose may be useful in the in vivo detection of eATP in murine models of skin diseases.

  11. 细胞外ATP和水杨酸对烟草光合指标的影响%Effects of extracellular ATP and salicylic acid on photosynthetic index of tobacco

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    冯汉青; 焦青松

    2014-01-01

    研究了细胞外ATP(eATP)和水杨酸(SA)对烟草(Nicotiana tabacum)叶片的气孔导度(GH2 O)、蒸腾速率(E)、光合作用速率(A)与叶绿素荧光参数[包括PSⅡ潜在最大光化学量子效率(Fv/Fm)、PSⅡ光适应下实际光化学效率Y(Ⅱ)、电子传递速率(ETR)、非光化学荧光淬灭(NPQ)和光化学荧光淬灭(qP)]的影响。结果表明:SA能导致A、GH2 O和E的下降,而eATP的处理能缓解SA造成的A、GH2 O和E的下降;但SA未对叶绿素荧光参数Fv/Fm、Y(Ⅱ)、NPQ、qP和ETR造成显著影响,eATP的加入也未改变SA处理下叶片叶绿素荧光参数的水平。这说明SA能导致光合作用的抑制,而eATP能明显缓解SA对光合作用的抑制,但以上作用可能均和光反应阶段无关。并对其内在机理进行了探讨。%The present work studied the effects of extracellular ATP (eATP )and salicylic acid (SA)on transpiration rate (E),stomatal conductance (GH2 O),photosynthesis rate (A)and chlorophyll fluorescence parameters,including maximal photochemical efficiency of PSⅡ(Fv/Fm),effective photochemical quantum yield of PSⅡ(Y(Ⅱ)),PSⅡelec-tron transport rate (ETR),non-photochemical quenching coefficient (NPQ)and the coefficient of photochemical fluo-rescence (qP))of tobacco (Nicotiana tabacum).The results showed that the transpiration rate (E),stomatal con-ductance (GH2 O)and photosynthesis rate (A)decreased under SA stress,while these decreases were anesised by the addition of eATP .However,SA had no significant effects on the chlorophyll fluorescence parameters,and the addition of eATP did not significantly changed the values of the chlorophyll fluorescence parameters under SA stress.These results indicated that SA could inhibit photosynthesis of tobacco leaves and the addition of eATP could alleviate the inhibition of photosynthesis by SA stress,while these effects were not related to the light reaction.We also discussed the possible mechanism based on these observations.

  12. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

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    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  13. Extracellular nucleotide signaling in plants

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    Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)

    2016-09-08

    Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

  14. A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems.

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    Vanegas, Diana C; Clark, Greg; Cannon, Ashley E; Roux, Stanley; Chaturvedi, Prachee; McLamore, Eric S

    2015-12-15

    The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8 nA/µM, a response time of 20±13 s and a lower detection limit of 1.3±0.7 nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment.

  15. Extracellular ATP acts as a damage associated molecular pattern (DAMP signal in plants

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    Kiwamu eTanaka

    2014-09-01

    Full Text Available As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs. ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling role in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor Kinase, which is plant-specific. P2K1 (DORN1 is required for ATP-induced cellular responses (e.g., cytosolic Ca2+ elevation, MAPK phosphorylation, and gene expression. Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of the future research of extracellular ATP as a DAMP signal.

  16. A receptor that is highly specific for extracellular ATP in developing chick skeletal muscle in vitro.

    OpenAIRE

    Thomas, S A; Zawisa, M. J.; X. Lin; Hume, R. I.

    1991-01-01

    1. Extracellular adenosine 5'-triphosphate (ATP) activated an early excitatory conductance followed by a late potassium conductance in developing chick skeletal muscle. A series of ATP analogues were tested for their ability to activate these two conductances. All compounds tested were either agonists for both responses or for neither. Furthermore, the potency of agonists was similar for the two responses. 2. The order of potency for agonists was ATP approximately adenosine 5'-O-(3-thiotripho...

  17. Extracellular ATP and adenosine : The Yin and Yang in immune responses?

    NARCIS (Netherlands)

    Faas, M. M.; Saez, T.; de Vos, P.

    Extracellular adenosine 50-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under

  18. Effects of Extracellular ATP and Its Receptors on Peripheral Nerve Regeneration in SD Rats

    Institute of Scientific and Technical Information of China (English)

    王栓科; 张致英; 洪光祥; 王同光; 王发斌; 康皓

    2003-01-01

    To explore the effect of the extracellular ATP and its receptors on axon regeneration in the sciatic nerve defect in rats, 0. 5 cm defect of the sciatic nerve was made and repaired with long arm silicon tube like a "Y" type. The single arm of the silicon tube was sutured to proximal of the sciatic nerve. 10μl 1 mmol/L ATP in physiological saline was injected into the left chamber of the silicon tube (experimental group) and physiological saline injected into another silicon tube as a control group. In another model 1 mmol/L 10 μl ATP and 1 mmol/L 10μl ATP+0.2 mg/ml suramin were injected respectively into two arms of the silicon tube. After 4 and 8 weeks the specimens were obtained from the silicon tube for examining the axon regeneration histologically and image analysis. All the regeneration axons grew into the silicon tube containing the ATP, but there was no axon regeneration in the silicon tube containing the ATP+Suramin or physiological saline. It was demonstrated that extracellular ATP had a powerful attraction to the regenerated axon of peripheral nerve. The suramin inhibited the axon induction of the extracellular ATP completely via the ATP receptors.

  19. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

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    Shin, Youn Ho; Lee, Seo Jin [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of); Jung, Junyang, E-mail: jjung@khu.ac.kr [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

  20. The P2X7 receptor is an important regulator of extracellular ATP levels

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    Andrea eBrandao-Burch

    2012-03-01

    Full Text Available Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signalling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells, possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05-0.5pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are 4-fold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1-100µM AZ10606120, A438079 and KN-62 were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079 and KN-62, at 0.1-10µM, decreased ATP release by mature osteoclasts by up to 70%, 60% and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1-100µM NEM or brefeldin A had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1-10µM also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signalling in bone.

  1. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

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    Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

    2016-01-01

    Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

  2. Regulation of extracellular ATP in human erythrocytes infected with Plasmodium falciparum.

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    Cora Lilia Alvarez

    Full Text Available In human erythrocytes (h-RBCs various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages. A "3V" mixture containing isoproterenol (β-adrenergic agonist, forskolin (adenylate kinase activator and papaverine (phosphodiesterase inhibitor was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs, t-RBCs (trophozoite-infected RBCs and s-RBCs (schizont-infected RBCs showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe] increased nonlinearly with parasitemia (from 2 to 12.5%. Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an

  3. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

    Directory of Open Access Journals (Sweden)

    M Florencia Leal Denis

    Full Text Available The peptide mastoparan 7 (MST7 triggered in human erythrocytes (rbcs the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe, interacting with P (purinergic receptors, can affect cell volume (Vr, we explored the dynamic regulation between Vr and ATPe.We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors.In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%.Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

  4. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank B; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco(2) showed negligible hemolysis (ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than...... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O(2) saturation did not lead to additional ATP release. An elevation of Pco(2) was also without influence on erythrocyte ATP release. In the saline...

  5. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than...... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco2 was also without influence on erythrocyte ATP release. In the saline...

  6. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than...... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco2 was also without influence on erythrocyte ATP release. In the saline...

  7. ATP, an extracellular signaling molecule in red blood cells: A messenger for malaria?

    Directory of Open Access Journals (Sweden)

    Ghania Ramdani

    2014-10-01

    Full Text Available Adenosine 5′ triphosphate (ATP, discovered in 1929 by Karl Lohmannest, is described as an essential energy source for cells. In the biochemistry of all living organisms, ATP hydrolysis provides the energy required for the chemical reactions of metabolism. It is the precursor of a number of essential enzyme cofactors, such as nicotinamide adenine dinucleotide (NAD + and coenzyme A [NAD + , flavin adenine dinucleotide (FAD, and is ATP coenzyme A are all formed from ATP] and is the source of the phosphoryl group in most kinase-mediated phosphorylation reactions. Another essential, but less known function is that ATP plays a very important role as an extracellular signaling molecule, allowing cells and tissues to communicate. ATP is converted into cAMP, a major second messenger involved in many cellular processes, by adenylyl cyclase, a membrane-associated enzyme. In this review, we describe the role of ATP as a beneficial extracellular molecule released by healthy red blood cells (RBCs in response to hypoxia to mediate a vasodilator signal, by oxidatively stressed RBCs, and by Plasmodium falciparum-infected RBCs (iRBCs, and its similarity with released ATP that by the combined action of the ectonucleotidases CD39 and CD73 is converted to adenosine that mediates sickling in sickle cell disease (SCD.

  8. Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection.

    Science.gov (United States)

    Séror, Claire; Melki, Marie-Thérèse; Subra, Frédéric; Raza, Syed Qasim; Bras, Marlène; Saïdi, Héla; Nardacci, Roberta; Voisin, Laurent; Paoletti, Audrey; Law, Frédéric; Martins, Isabelle; Amendola, Alessandra; Abdul-Sater, Ali A; Ciccosanti, Fabiola; Delelis, Olivier; Niedergang, Florence; Thierry, Sylvain; Said-Sadier, Najwane; Lamaze, Christophe; Métivier, Didier; Estaquier, Jérome; Fimia, Gian Maria; Falasca, Laura; Casetti, Rita; Modjtahedi, Nazanine; Kanellopoulos, Jean; Mouscadet, Jean-François; Ojcius, David M; Piacentini, Mauro; Gougeon, Marie-Lise; Kroemer, Guido; Perfettini, Jean-Luc

    2011-08-29

    Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches. © 2011 Séror et al.

  9. Extracellular Adenosine Triphosphate Associated with Amphibian Erythrocytes: Inhibition of ATP Release by Anion Channel Blockers.

    Science.gov (United States)

    1986-01-01

    ATP may mediate contraction in the urinary bladder of the rat and guinea-pig (53,63,99,238), relaxation in taenia coli (17,63,87,173,380,381) and...receptors. This uncertainty has been generated because of findings in rabbit anococcygeus muscle (405) and guinea-pig taenia coli (457), in which, as...and Holmberg, B. The effects of extracellularly -~ applied ATP and related compounds on electrical and mechanical activity of the smooth muscle taenia

  10. ATP release and purinergic signaling in NLRP3 inflammasome activation

    Directory of Open Access Journals (Sweden)

    Isabelle eCOUILLIN

    2013-01-01

    Full Text Available The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns. One step- or two step- models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers ATP release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP (eATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key proinflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

  11. Effects of Extracellular ATP on Survival of Sensory Neurons in the Dorsal Root Ganglia of Rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    ATP was added to the cultured sensory neurons obtained from the dorsal root ganglia of the neonatal rats and PBS was added to serve as control. MTT assays were conducted to evaluate the survival and activity of the cultured neurons. And the silicone regenerative chamber was used after the sciatic nerve incision of the mature SD rat. 1 mmol/L ATP was injected into the left chamber and 0.09 % natrium chloride was injected into the right chamber as controls. The changes of nitric oxide synthase (NOS) activity in the corresponding dorsal root ganglia were measured histochemically and image analysis was also performed 4 days after the sciatic nerve injury. The results showed that extracellular ATP could enhance the survival of the neurons and the number of NOS positive neurons were significantly different between the ATP and control groups (P<0.05). It was suggested that extracellular ATP had neurotrophic effect on neurons survival and could inhibit the NOS activity of the sensory neurons after the peripheral nerve incision, hence exerting the protective effect on the neurons, which was valuable for nerve regeneration after nerve injury.

  12. Extracellular ATP Promotes Stomatal Opening of Arabidopsis thaliana through Heterotrimeric G Protein Subunit and Reactive Oxygen Species

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hao; Wei-Xia Wang; Chen Chen; Yu-Fang Wang; Ting Liu; Xia Li; Zhong-Lin Shang

    2012-01-01

    In recent years,adenosine tri-phosphate(ATP)has been reported to exist in apoplasts of plant cells as a signal molecule.Extracellular ATP(eATP)plays important roles in plant growth,development,and stress tolerance.Here,extracellular ATP was found to promote stomatal opening of Arabidopsis thaliana in light and darkness.ADP,GTP,and weakly hydrolyzable ATP analogs(ATPγS,Bz-ATP,and 2meATP)showed similar effects,whereas AMP and adenosine did not affect stomatal movement.Apyrase inhibited stomatal opening.ATP-promoted stomatal opening was blocked by an NADPH oxidase inhibitor(diphenylene iodonium)or deoxidizer(dithiothreitol),and was impaired in null mutant of NADPH oxidase(atrbohD/F).Added ATP triggered ROS generation in guard cells via NADPH oxidase.ATP also induced Ca2+ influx and H+ efflux in guard cells.In atrbohD/F,ATP-induced ion flux was strongly suppressed.In null mutants of the heterotrimeric G protein α subunit,ATP-promoted stomatal opening,cytoplasmic ROS generation,Ca2+ influx,and H+ efflux were all suppressed.These results indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein,ROS,cytosolic Ca2+,and plasma membrane H+-ATPase.

  13. Extracellular ATP in the lymphohematopoietic system: P2Z purinoceptors and membrane permeabilization

    Directory of Open Access Journals (Sweden)

    Persechini P.M.

    1998-01-01

    Full Text Available The effects of extracellular nucleosides and nucleotides on many organs and systems have been recognized for almost 50 years. The effects of extracellular ATP (ATPo, UTPo, ADPo, and other agonists are mediated by P2 purinoceptors. One of the most dramatic effects of ATPo is the permeabilization of plasma membranes to low molecular mass solutes of up to 900 Da. This effect is evident in several cells of the lymphohematopoietic system and is supposed to be mediated by P2Z, an ATP4--activated purinoceptor. Here, we review some basic information concerning P2 purinoceptors and focus our attention on P2Z-associated phenomena displayed by macrophages. Using fluorescent dye uptake, measurement of free intracellular Ca2+ concentration and electrophysiological recordings, we elucidate some of the events that follow the application of ATP to the extracellular surface of macrophages. We propose a regulatory mechanism for the P2Z-associated permeabilization pore. The presence of P2 purinoceptors in cells of the lymphohematopoietic system makes them potential candidates to mediate immunoregulatory events

  14. Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats

    NARCIS (Netherlands)

    Spaans, F.; Melgert, B. N.; Chiang, C.; Borghuis, T.; Klok, P. A.; de Vos, P.; van Goor, H.; Bakker, W.W.; Faas, M. M.

    2014-01-01

    Introduction: Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant

  15. Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease

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    Leigh A. Swayne

    2017-08-01

    Full Text Available Pannexin 1 (Panx1 channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs. Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

  16. Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

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    Rozan Vroman

    2014-05-01

    Full Text Available Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms, highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²⁺ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form

  17. Extracellular ATP and other nucleotides-ubiquitous triggers of intercellular messenger release.

    Science.gov (United States)

    Zimmermann, Herbert

    2016-03-01

    Extracellular nucleotides, and ATP in particular, are cellular signal substances involved in the control of numerous (patho)physiological mechanisms. They provoke nucleotide receptor-mediated mechanisms in select target cells. But nucleotides can considerably expand their range of action. They function as primary messengers in intercellular communication by stimulating the release of other extracellular messenger substances. These in turn activate additional cellular mechanisms through their own receptors. While this applies also to other extracellular messengers, its omnipresence in the vertebrate organism is an outstanding feature of nucleotide signaling. Intercellular messenger substances released by nucleotides include neurotransmitters, hormones, growth factors, a considerable variety of other proteins including enzymes, numerous cytokines, lipid mediators, nitric oxide, and reactive oxygen species. Moreover, nucleotides activate or co-activate growth factor receptors. In the case of hormone release, the initially paracrine or autocrine nucleotide-mediated signal spreads through to the entire organism. The examples highlighted in this commentary suggest that acting as ubiquitous triggers of intercellular messenger release is one of the major functional roles of extracellular nucleotides. While initiation of messenger release by nucleotides has been unraveled in many contexts, it may have been overlooked in others. It can be anticipated that additional nucleotide-driven messenger functions will be uncovered with relevance for both understanding physiology and development of therapy.

  18. Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Hein, Martina; Petersen, Frank; Thon, Lutz; Adam, Dieter; Bulfone-Paus, Silvia

    2005-04-01

    Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

  19. The expression and significance of extracellular ATP in murine acute liver injury model%胞外ATP在小鼠急性肝损伤中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    胡梅琮; 邹玲莉; 黄保军; 王磊

    2014-01-01

    Objective To observe the expression and significance of extracellular adenosine triphosphate (eATP) in concanavalin A (ConA)-induced murine acute liver injury model.Methods A total of 72 mice were randomly classified into control group (saline,36 cases)and ConA group (20 mg/kg ConA,36 cases).The blood specimens and liver tissues were collected at 2,6,12,18,24 and 48 h after injection.The activities of serum alanine aminotransferase (ALT)were measured by Reitman Frankel assay.Hematoxylin-eosin (HE)dyeing was carried out to assess the pathological change of liver tissue.The levels of eATP in serum were detected by chemiluminescence. Western-blot was employed to detect the expression of purinoceptor P2(P2X7).The contents of serum interleukin 1 beta (IL-1β)were assayed by enzyme-linked immunosorbent assay (ELISA).Results The ConA-induced murine acute liver injury model was constructed successfully.The level of eATP increased at 2 h after ConA injection,and reached peak at 18 h (700 nmol /L).Meanwhile,there expressed P2X7 in liver tissues.Compared with control group,the IL-1βlevels in serum of ConA group increased significantly (P<0.01).Conclusions In ConA-induced murine acute liver injury model,eATP releases from the injury liver tissues,and might influence the synthesis and secretion of inflammatory cytokine IL-1βthrough the P2X7 pathway,eventually aggravating the process of acute liver injury.%目的:探讨胞外三磷酸腺苷(eATP)在刀豆蛋白A(ConA)诱导小鼠急性肝损伤中的表达及意义。方法将72只昆明种小鼠随机分为对照组(36只)、ConA组(36只)。ConA组由尾静脉处注射20 mg/kg ConA,对照组注射同体积的无致热原生理盐水。2组分别于注射后2、6、12、18、24、48 h留取血液标本和肝脏标本。采用赖氏法检测血清丙氨酸氨基转移酶(ALT)活性,苏木素-伊红(HE )染色法检查肝组织病理学改变,化学发光技术检测血清eATP水平,免疫印

  20. Trophic actions of extracellular ATP: gene expression profiling by DNA array analysis.

    Science.gov (United States)

    Neary, J T

    2000-07-01

    In addition to Professor Burnstock's work on the short-term signaling actions of extracellular nucleotides and nucleosides, Geoff has had a long-standing interest in trophic actions of purines in development and in pathophysiological conditions which has been instrumental in encouraging my work in this area. The trophic actions of extracellular ATP, alone or in combination with polypeptide growth factors, may play an important role in brain development and may contribute to the reactive gliosis that accompanies brain injury and neurodegeneration. P2Y receptors in astrocytes are coupled to the ERK/MAPK cascade, a signal transduction mechanism crucial for cellular proliferation and differentiation. The mitogenic signaling pathway from P2Y receptors to ERK involves phospholipase D and a calcium-independent PKC isoform, PKCdelta. DNA array analysis reveals a number of changes in gene expression after P2Y receptor occupancy, indicating that this methodology will be a powerful tool in understanding the mechanisms underlying the trophic actions of extracellular nucleotides and nucleosides.

  1. Extracellular ATP mediates necrotic cell swelling in SN4741 dopaminergic neurons through P2X7 receptors.

    Science.gov (United States)

    Jun, Dong-Jae; Kim, Jaeyoon; Jung, Sang-Yong; Song, Ran; Noh, Ji-Hyun; Park, Yong-Soo; Ryu, Sung-Ho; Kim, Joung-Hun; Kong, Young-Yun; Chung, Jun-Mo; Kim, Kyong-Tai

    2007-12-28

    Extracellular ATP has recently been identified as an important regulator of cell death in response to pathological insults. When SN4741 cells, which are dopaminergic neurons derived from the substantia nigra of transgenic mouse embryos, are exposed to ATP, cell death occurs. This cell death is associated with prominent cell swelling, loss of ER integrity, the formation of many large cytoplasmic vacuoles, and subsequent cytolysis and DNA release. In addition, the cleavage of caspase-3, a hallmark of apoptosis, is induced by ATP treatment. However, caspase inhibitors do not overcome ATP-induced cell death, indicating that both necrosis and apoptosis are associated with ATP-induced cell death and suggesting that a necrotic event might override the apoptotic process. In this study we also found that P2X(7) receptors (P2X(7)Rs) are abundantly expressed in SN4741 cells, and both ATP-induced swelling and cell death are reversed by pretreatment with the P2X(7)Rs antagonist, KN62, or by knock-down of P2X(7)Rs with small interfering RNAs. Therefore, extracellular ATP release from injured tissues may act as an accelerating factor in necrotic SN4741 dopaminergic cell death via P2X(7)Rs.

  2. Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

    Directory of Open Access Journals (Sweden)

    Becker Walter

    2011-09-01

    Full Text Available Abstract Background Treatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells. We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells. Results The activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM. UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS. This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin and BDNF (brain derived neurotrophic factor. Conclusion The present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein

  3. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

    Science.gov (United States)

    Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

    2017-01-01

    Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

  4. Inflammasome activation in bovine monocytes by extracellular ATP does not require the purinergic receptor P2X7.

    Science.gov (United States)

    Hussen, Jamal; Düvel, Anna; Koy, Mirja; Schuberth, Hans-Joachim

    2012-10-01

    Extracellular adenosine triphosphate (ATP) is a second signal for the assembly of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, which form a framework to activate caspase 1, leading to the processing and secretion of the pro-inflammatory cytokine interleukin-1β (IL-1β). The aim of the present study was to investigate the role of the ATP-gated ion channel subtype P2X7 receptor in the inflammasome activation of bovine monocytes. ATP-induced inflammasome assembly in bovine monocytes was shown by caspase-1 activation and the release of IL-1β by LPS/ATP-stimulated bovine cells. The IL-1β release depended on potassium efflux but was independent of reactive oxygen generation of bovine monocytes. Unlike in the human system, a P2X7 receptor antagonist did not block the ATP-induced release of IL-1β of LPS-primed bovine cells. P2X7 mediated pore formation was observed in subsets of bovine T lymphocytes (CD4+>CD8+) but not in monocytes. In addition, ATP and 2-MeSATP but not the high affinity P2X7 agonist BzATP induced calcium influx in bovine monocytes. The data indicate that ROS generation plays no role in the ATP-induced activation of inflammasome in bovine monocytes and that P2X7-mediated pore formation is not necessary for the release of Interleukin-1β.

  5. Modulation of Extracellular ATP Content of Mast Cells and DRG Neurons by Irradiation: Studies on Underlying Mechanism of Low-Level-Laser Therapy

    Directory of Open Access Journals (Sweden)

    Lina Wang

    2015-01-01

    Full Text Available Low-level-laser therapy (LLLT is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca2+]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

  6. Dynamic increase in extracellular ATP accelerates photoreceptor cell apoptosis via ligation of P2RX7 in subretinal hemorrhage.

    Directory of Open Access Journals (Sweden)

    Shoji Notomi

    Full Text Available Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD. In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV, growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor. Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG, a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.

  7. Direct excitation of inhibitory interneurons by extracellular ATP mediated by P2Y1 receptors in the hippocampal slice.

    Science.gov (United States)

    Kawamura, Masahito; Gachet, Christian; Inoue, Kazuhide; Kato, Fusao

    2004-12-01

    ATP is an important cell-to-cell signaling molecule mediating the interactions between astrocytes and neurons in the CNS. In the hippocampal slices, ATP suppresses excitatory transmission mostly through activation of adenosine A1 receptors, because the ectoenzyme activity for the extracellular breakdown of ATP to adenosine is high in slice preparations in contrast to culture environments. Because the hippocampus is also rich in the expression of P2 receptors activated specifically by ATP, we examined whether ATP modulates neuronal excitability in the acute slice preparations independently of adenosine receptors. Although ATP decreased the frequency of spontaneously occurring EPSCs in the CA3 pyramidal neurons through activation of adenosine A1 receptors, ATP concurrently increased the frequency of IPSCs in a manner dependent on action potential generation. This effect was mediated by P2Y1 receptors because (1) 2-methylthio-ATP (2meSATP) was the most potent agonist, (2) 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate diammonium (MRS2179) abolished this effect, and (3) this increase in IPSC frequency was not observed in the transgenic mice lacking P2Y1 receptor proteins. Application of 2meSATP elicited MRS2179-sensitive time- and voltage-dependent inward currents in the interneurons, which depolarized the cell to firing threshold. Also, it increased [Ca2+]i in both astrocytes and interneurons, but, unlike the former effect, the latter was entirely dependent on Ca2+ entry. Thus, in hippocampal slices, in addition to activating A1 receptors of the excitatory terminals after being converted to adenosine, ATP activates P2Y1 receptors in the interneurons, which is linked to activation of unidentified excitatory conductance, through mechanisms distinct from those in the astrocytes.

  8. [Identification of a new pro-invasion factor in tumor microenvironment: progress in function and mechanism of extracellular ATP].

    Science.gov (United States)

    Fang, W G; Tian, X X

    2017-04-18

    Up to 90% of all cancer related morbidity and mortality can be attributed to metastasis. In recent years the study of tumor microenvironment, its cellular and molecular components, and how they can affect neoplastic progression toward metastasis, has become a hot focus in cancer research. Accumulated evidence shows that the formation of metastasis is a multi-step sequential process, in which, the tumor cells continuously interact with the host microenvironment. Host derived factors, i.e. growth factors/inhibitors, angiogenic factors, chemokines, etc. together with different types of host cells, play important roles in the tumor progression towards metastasis. The interaction between the tumor cells and host microenvironment determines the fate of metastasis. The reveal of this interaction mechanism provides us an opportunity to find effective mode of interference and develop novel anti-metastasis drugs. In this review, we have summarized our work on a new pro-invasion factor identified in tumor microenvironment and how it affects tumor invasion and metastass. Adenosine triphosphate (ATP), the key intracellular energy currency, accumulates within the tumor microenvironment and is closely involved in cancer cell metabolism and in antitumor immunity. The established role of ATP as a growth modulator and a proinflammatory mediator endues ATP and other purines with potential players in host-tumor interaction. Our study demonstrated that extracellular ATP stimulated human cancer invasion in in vitro tests. Increased migration and invasive ability across Matrigel was observed in some human carcinoma cell lines, including the prostate, breast, colon, melanoma and lung, when stimulated with ATP or its analogues. ATP enhanced the motility of cancer cells via increasing the amount and length of lamellipodia and filopodia, which were necessary for the cell motility. Significant increase in Rac1 and Cdc42 activities was observed. Using cDNA microarray we found that the

  9. Release of ATP from marginal cells in the cochlea of neonatal rats can be induced by changes in extracellular and intracellular ion concentrations.

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    Yating Peng

    Full Text Available BACKGROUND: Adenosine triphosphate (ATP plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. METHODS: Sprague-Dawley rats aged 1-3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. RESULTS: Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K(+ and intra- and extracellular Ca(2+. Furthermore, changes in the concentration of intracellular Ca(2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. CONCLUSION: We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K(+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A(2.

  10. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Icyuz, Mert; Chauvet, Sylvain; Tao, Binli; Hartman, John L; Kirk, Kevin L

    2016-03-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.

  11. T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP

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    Lisa Perruzza

    2017-03-01

    Full Text Available The ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh cell abundance in the Peyer’s patches (PPs of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help.

  12. Production of adenosine from extracellular ATP at the striatal cholinergic synapse.

    Science.gov (United States)

    James, S; Richardson, P J

    1993-01-01

    The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC 3.6.1.15) had a Km of 131 microM, whereas the ecto-ADPase (EC 3.6.1.6) had a Km of 58 microM, was Ca(2+)-dependent, and was inhibited by the ATP analogue 5'-adenylylimidodiphosphate (AMPPNP). The ecto-5'-nucleotidase (EC 3.1.3.5) had a Km of 21 microM, was inhibited by AMPPNP and alpha,beta-methylene ADP, and by a specific antiserum. The Vmax values of the ATPase, ADPase, and 5'-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5'-nucleotidase enzyme, which amounted to 40% of the total 5'-nucleotidase activity, was inhibited by AMPPNP, alpha,beta-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5'-nucleotidase, and was inversely proportional to the ecto-5'-nucleotidase activity. The function and characteristics of this pathway and the central role of 5'-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.

  13. The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction.

    Science.gov (United States)

    Giniatullin, A; Petrov, A; Giniatullin, R

    2015-01-29

    Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular

  14. Extracellular ATP induces the release of calcium from intracellular stores without the activation of protein kinase C in Swiss 3T6 mouse fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez, F.A.; Rozengurt, E.; Heppel, L.A. (Cornell Univ., Ithaca, NY (USA))

    1989-06-01

    Exposure of Swiss 3T6 mouse fibroblasts to extracellular ATP stimulated the formation of inositol phosphates and mobilized intracellular calcium. The mobilization of intracellular calcium was verified by imaging of fura-2 fluorescence in individual cells and by monitoring the efflux of {sup 45}Ca{sup 2+} from preloaded cells. However, the authors found no activation of protein kinase C as measured by phosphorylation of an 80-kDa acidic protein and by transmodulation of the receptor for epidermal growth factor. A careful examination of the kinetics of the phosphorylation reaction (from 30 sec to 10 min) revealed no activation of protein kinase C by extracellular ATP at any time. The lack of activation of protein kinase C was demonstrated even when a concentration of ATP 10-fold higher than that required to give a strong Ca{sup 2+} signal was used. Extracellular ATP did not inhibit protein kinase C activation by fetal bovine serum, platelet-derived growth factor, or phorbol esters. The effects of ATP were also produced by UTP but not by ADP, AMP, or adenosine. These findings demonstrate that it is possible to induce the mobilization of intracellular calcium by an inositol phosphate-mediated pathway without the activation of protein kinase C.

  15. Effect of extracellular adenosine 5'-triphosphate on cryopreserved epididymal cat sperm intracellular ATP concentration, sperm quality, and in vitro fertilizing ability.

    Science.gov (United States)

    Thuwanut, Paweena; Arya, Nlin; Comizzoli, Pierre; Chatdarong, Kaywalee

    2015-09-15

    Intracellular adenosine 5'-triphosphate (ATP) is essential for supporting sperm function in the fertilization process. During cryopreservation, damage of sperm mitochondrial membrane usually leads to compromised production of intracellular ATP. Recently, extracellular ATP (ATPe) was introduced as a potent activator of sperm motility and fertilizing ability. This study aimed to evaluate (1) levels of intracellular ATP in frozen-thawed epididymal cat sperm after incubation with ATPe and (2) effects of ATPe on epididymal cat sperm parameters after freezing and thawing. Eighteen male cats were included. For each replicate, epididymal sperm from two cats were pooled to one sample (N = 9). Each pooled sample was cryopreserved with the Tris-egg yolk extender into three straws. After thawing, the first and second straws were incubated with 0-, 1.0-, or 2.5-mM ATPe for 10 minutes and evaluated for sperm quality at 10 minutes, 1, 3, and 6 hours after thawing and fertilizing ability. The third straw was evaluated for intracellular ATP concentration in control and with 2.5-mM ATPe treatment. Higher concentration of intracellular sperm ATP was observed in the samples treated with 2.5-mM ATPe compared to the controls (0.339 ± 0.06 μg/2 × 10(6) sperm vs. 0.002 ± 0.003 μg/2 × 10(6) sperm, P ≤ 0.05). In addition, incubation with 2.5-mM ATPe for 10 minutes promoted sperm motility (56.7 ± 5.0 vs. 53.3 ± 4.4%, P ≤ 0.05) and progressive motility (3.1 ± 0.2 vs. 2.8 ± 0.4, P ≤ 0.05), mitochondrial membrane potential (36.4 ± 5.5 vs. 28.7 ± 4.8%, P ≤ 0.05), and blastocyst rate (36.1 ± 7.0 and 28.8 ± 7.4%, P ≤ 0.05) compared with the controls. In contrast, ATPe remarkably interfered acrosome integrity after 6 hours of postthawed incubation. In sum, the present finding that optimal incubation time of postthaw epididymal cat sperm under proper ATPe condition might constitute a rationale for the studies on other endangered wild felids regarding sperm quality and embryo

  16. Growth inhibitory effect and apoptosis induced by extracellular ATP and adenosine on human gastric carcinoma cells: involvement of intracellular uptake of adenosine

    Institute of Scientific and Technical Information of China (English)

    Ming-xia WANG; Lei-ming REN

    2006-01-01

    receptors. Conclusion: Extracellular ATP and ADO reduced the cell viability, arrested cell cycle and induced apoptosis in HGC-27 cell line by intracellular uptake of ADO. One of the main routes of ATP-induced apoptosis in HGC-27 cells is through the breakdown to adenosine.

  17. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou (NCSU)

    2016-10-26

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  18. Extracellular vesicles released from mesenchymal stromal cells modulate miRNA in renal tubular cells and inhibit ATP depletion injury.

    Science.gov (United States)

    Lindoso, Rafael S; Collino, Federica; Bruno, Stefania; Araujo, Dayana S; Sant'Anna, Julliana F; Tetta, Ciro; Provero, Paolo; Quesenberry, Peter J; Vieyra, Adalberto; Einicker-Lamas, Marcelo; Camussi, Giovanni

    2014-08-01

    The mechanisms involved in renal repair by mesenchymal stromal cells (MSCs) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSCs have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells through the transference of several molecules, like protein, bioactive lipids, mRNA, and microRNAs (miRNAs). The miRNAs are noncoding RNAs that posttranscriptionally modulate gene expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTECs with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTECs. Prediction of miRNA targets showed that miRNAs modulated in PTECs are involved in process of renal recovery with downregulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization, and hypoxia, such as CASP3 and 7, SHC1 and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTECs.

  19. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...... that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca2+ but, despite subsequent spikes in Ca2+ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes...

  20. Extracellular ATP does not induce P2X7 receptor-dependent responses in cultured renal- and liver-derived swine macrophages

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    Takato Takenouchi

    2014-01-01

    Full Text Available The P2X7 receptor (P2X7R is an ATP-gated cation channel that is abundantly expressed in monocytes/macrophages. P2X7R activation by ATP results in various cellular responses including Ca2+ influx, membrane pore formation, and cytokine secretion. Since P2X7R has low affinity for ATP, high concentrations of ATP (in the mM range are generally required to activate this receptor in vitro. Functional expression of P2X7R has been detected in monocytes/macrophages obtained from different animal species including humans, rodents, dogs, and bovines, but so far it has not been detected in swine (Sus scrofa. In this study, we investigated the expression and functions of P2X7R in swine macrophages, which were isolated from mixed primary cultures of swine kidney or liver tissue. The P2X7R mRNA and protein expression observed in the swine macrophages was comparable to that seen in a c-myc-immortalized mouse kidney-derived clonal macrophage cell line (KM-1. However, extracellular ATP did not induce P2X7R-dependent sustained Ca2+ influx, membrane pore formation, or the secretion of the bioactive cytokine interleukin-1β in the swine macrophages, whereas these responses were clearly observed in the mouse KM-1 cells after stimulation with millimolar concentrations of ATP as a positive control. These findings suggest that the ATP/P2X7R pathway is impaired in swine macrophages at least in the culture conditions used in the present study.

  1. Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca(2+) concentration, we simultaneously measured Ca(2+) oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  2. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...

  3. A possible role for extra-cellular ATP in plant responses to high frequency, low amplitude electromagnetic field.

    Science.gov (United States)

    Roux, David; Faure, Catherine; Bonnet, Pierre; Girard, Sébastien; Ledoigt, Gérard; Davies, Eric; Gendraud, Michel; Paladian, Françoise; Vian, Alain

    2008-06-01

    In parallel to evoking the accumulation of stress-related transcripts, exposure to low level 900 MHz EMF affected the levels of ATP, the main energy molecule of the cell. Its concentration dropped rapidly (27% after 30 min) in response to EMF exposure, along with a 18% decrease in the adenylate energy charge (AEC), a good marker of cell energy status. One could interpret this decrease in ATP and AEC in a classical way, i.e., as the result of an increase in cellular energy usage, but recent work brings exciting new insights in pointing out a signalling function for ATP, especially in the stress physiology context where it could trigger both reactive oxygen species and calcium movement (this latter being involved in plant responses to EMF exposure). In this addendum, we discuss our results within this new perspective for ATP function.

  4. Altered extracellular ATP, ADP, and AMP hydrolysis in blood serum of sedentary individuals after an acute, aerobic, moderate exercise session.

    Science.gov (United States)

    Moritz, Cesar Eduardo Jacintho; Teixeira, Bruno Costa; Rockenbach, Liliana; Reischak-Oliveira, Alvaro; Casali, Emerson André; Battastini, Ana Maria Oliveira

    2017-02-01

    Nucleotidases participate in the regulation of physiological and pathological events, such as inflammation and coagulation. Exercise promotes distinct adaptations, and can influence purinergic signaling. In the present study, we investigated soluble nucleotidase activities in the blood serum of sedentary young male adults at pre- and post-acute moderate aerobic exercise. In addition, we evaluated how this kind of exercise could influence adenine nucleotide concentrations in the blood serum. Sedentary individuals were submitted to moderate aerobic exercise on a treadmill; blood samples were collected pre- and post-exercise, and serum was separated for analysis. Results showed increases in ATP, ADP, and AMP hydrolysis post-exercise, compared to pre-exercise values. The ecto-nucleotide pyrophosphatase/phosphodiesterase was also evaluated, showing an increased activity post-exercise, compared to pre-exercise. Purine levels were analyzed by HPLC in the blood serum, pre- and post-exercise. Decreased levels of ATP and ADP were found post-exercise, in contrast with pre-exercise values. Conversely, post-exercise levels of adenosine and inosine increased compared to pre-exercise levels. Our results indicate an influence of acute exercise on ATP metabolism, modifying enzymatic behavior to promote a protective biological environment.

  5. Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette transporters gene enrichment in typhoid fever-infected Nigerian children

    Directory of Open Access Journals (Sweden)

    Resau James H

    2011-09-01

    bacterial invasion. Distinct gene expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We found novel down-regulation of ABC (ATP-binding cassette transporters genes such as ABCA7, ABCC5, and ABCD4 and ATPase activity as the highest enriched pathway. Conclusions We identified unique extracellular components and ABC transporters gene enrichments in typhoid fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for typhoid fever in Africa.

  6. Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells

    Science.gov (United States)

    Salvestrini, Valentina; Orecchioni, Stefania; Talarico, Giovanna; Reggiani, Francesca; Mazzetti, Cristina; Bertolini, Francesco; Orioli, Elisa; Adinolfi, Elena; Virgilio, Francesco Di; Pezzi, Annalisa; Cavo, Michele

    2017-01-01

    Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2×7R is the most consistently expressed by tumors. P2×7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2×7R. Here, we show that P2×7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2×7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo. Overall, our results demonstrate that P2×7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML. PMID:27980223

  7. ATP signals

    DEFF Research Database (Denmark)

    Novak, Ivana

    2016-01-01

    The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas......The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas...

  8. Is the extracellular ATP a key in X-linked Charcot-Marie-Tooth disease and in inherited non-syndromic deafness?

    OpenAIRE

    Mas del Molino, Ezequiel

    2011-01-01

    [spa] El ATP es una molécula ampliamente conocida por su papel en muchas funciones como la homeostasis celular, el mantenimiento de gradientes iónicos, el mantenimiento del pH en gránulos secretores, el almacenamiento energético, regulador de la interacción actina-miosina, etc. Además, el ATP puede actuar como molécula señalizadora a través de los receptores purinérgicos P2. De receptores P2 hay de dos tipos, los P2X, que son ionotrópicos, y los P2Y que son metabotrópicos. Los primeros son un...

  9. Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.

    Science.gov (United States)

    Jiang, Shuang; Zhang, Yu; Zheng, Jin-Hua; Li, Xia; Yao, You-Li; Wu, Yan-Ling; Song, Shun-Zong; Sun, Peng; Nan, Ji-Xing; Lian, Li-Hua

    2017-03-01

    Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1

  10. Optogenetic control of ATP release

    Science.gov (United States)

    Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

    2013-03-01

    Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

  11. ATP as a signaling molecule: the exocrine focus

    DEFF Research Database (Denmark)

    Novak, Ivana

    2003-01-01

    Why and how do cells release ATP? It is not spilled energy. ATP becomes an extracellular regulator. Various cellular responses are initiated by purinergic receptors and signaling processes and are terminated by breakdown of ATP by ectonucleotidases. In epithelia, ATP regulates salt and water...

  12. ATP as a signaling molecule: the exocrine focus

    DEFF Research Database (Denmark)

    Novak, Ivana

    2003-01-01

    Why and how do cells release ATP? It is not spilled energy. ATP becomes an extracellular regulator. Various cellular responses are initiated by purinergic receptors and signaling processes and are terminated by breakdown of ATP by ectonucleotidases. In epithelia, ATP regulates salt and water tran...

  13. The involvement of nitric oxide in eATP-inhibited pollen germination and tube growth of Paulownia tomentosa%一氧化氮参与胞外ATP抑制泡桐花粉萌发和花粉管伸长

    Institute of Scientific and Technical Information of China (English)

    张媛华; 张韶杰; 佘小平

    2011-01-01

    The role of eATP and its relationship with nitric oxide(NO) in pollen germination and tube growth of Paulownia tomentosa were studied.The results showed that both eATP and NO donors(sodium nitroprusside) inhibited pollen germination and tube growth in a dose-dependence manner.Both NOS inhibitor(L-NAME) and NO scavenger(c-PTIO) partly reversed eATP-inhibited pollen germination and tube growth,indicating that the inhibitory effects of eATP on pollen germination and tube growth are related to NO generated by a NOS-like enzyme in pollen.%研究了胞外ATP(eATP)对泡桐(Paulownia tomaentosa Steud.)花粉萌发和花粉管伸长的效应及其与一氧化氮(NO)的关系.结果表明,eATP和NO供体硝普钠(SNP)均显著抑制花粉萌发和花粉管伸长,且表现明显浓度和时间依赖效应.一氧化氮合酶(NOS)抑制剂(L-NAME)和NO清除剂(c-PTIO)能够部分阻止eATP对花粉萌发和花粉管伸长的抑制作用,显示eATP抑制花粉萌发和花粉管伸长与其诱导NO产生有关,eATP诱导的NO可能由NOS催化产生.

  14. NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV Nonstructural Protein 5A (NS5A via MEK/Extracellular Signal Regulated Kinase (ERK Pathway

    Directory of Open Access Journals (Sweden)

    Xuesong Gao

    2013-05-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein 5A (NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.

  15. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    Science.gov (United States)

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  16. Real-time luminescence imaging of cellular ATP release.

    Science.gov (United States)

    Furuya, Kishio; Sokabe, Masahiro; Grygorczyk, Ryszard

    2014-03-15

    Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin-luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin-luciferase luminescence at ∼10 frames/s, sufficient to follow rapid ATP release with sensitivity of ∼10 nM and dynamic range up to 100 μM. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility.

  17. Detecting ATP release by a biosensor method.

    Science.gov (United States)

    Hayashi, Seiji; Hazama, Akihiro; Dutta, Amal K; Sabirov, Ravshan Z; Okada, Yasunobu

    2004-11-09

    Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

  18. Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

    Science.gov (United States)

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

    Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems.

  19. The Role of ATP in the Regulation of NCAM Function

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a direct...... ATP-NCAM interaction and discuss its functional implications. The ectodomain of NCAM contains the ATP binding Walker motif A and has intrinsic ATPase activity, which could modulate NCAM-dependent signaling processes. NCAM interacts directly with and signals through FGFR. The NCAM binding site to ATP...... overlaps with the site of NCAM-FGFR interaction, and ATP is capable of disrupting NCAM-FGFR binding. This implies that NCAM signaling through FGFR can be regulated by ATP, which is supported by the observation that ATP can abrogate NCAM-induced neurite outgrowth. Finally, ATP can induce NCAM ectodomain...

  20. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our...... aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan......-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient...

  1. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels.

    Science.gov (United States)

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-10-15

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K(+) (K(ATP)) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP](c)) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of K(ATP) channels in GR neurons, glucose (3 or 15 mM) caused no detectable increase in [ATP](c), monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25-luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mM) with a significant increase in [ATP](c). Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mM glucose with increases in [ATP](c). Further implicating an ATP-independent mechanism of K(ATP) channel closure in hypothalamic neurons, removal of extracellular glucose (10 mM) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mM) of intracellular ATP. Neurons from both brain regions responded to 5 mM lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP](c). High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed K(ATP) channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells.

  2. Lipoapoptosis induced by saturated free fatty acids stimulates monocyte migration: a novel role for Pannexin1 in liver cells.

    Science.gov (United States)

    Xiao, Feng; Waldrop, Shar L; Bronk, Steve F; Gores, Gregory J; Davis, Laurie S; Kilic, Gordan

    2015-09-01

    Recruitment of monocytes in the liver is a key pathogenic feature of hepatic inflammation in nonalcoholic steatohepatitis (NASH), but the mechanisms involved are poorly understood. Here, we studied migration of human monocytes in response to supernatants obtained from liver cells after inducing lipoapoptosis with saturated free fatty acids (FFA). Lipoapoptotic supernatants stimulated monocyte migration with the magnitude similar to a monocyte chemoattractant protein, CCL2 (MCP-1). Inhibition of c-Jun NH2-terminal kinase (JNK) in liver cells with SP600125 blocked migration of monocytes in a dose-dependent manner, indicating that JNK stimulates release of chemoattractants in lipoapoptosis. Notably, treatment of supernatants with Apyrase to remove ATP potently inhibited migration of THP-1 monocytes and partially blocked migration of primary human monocytes. Inhibition of the CCL2 receptor (CCR2) on THP-1 monocytes with RS102895, a specific CCR2 inhibitor, did not block migration induced by lipoapoptotic supernatants. Consistent with these findings, lipoapoptosis stimulated pathophysiological extracellular ATP (eATP) release that increased supernatant eATP concentration from 5 to ~60 nM. Importantly, inhibition of Panx1 expression in liver cells with short hairpin RNA (shRNA) decreased supernatant eATP concentration and inhibited monocyte migration, indicating that monocyte migration is mediated in part by Panx1-dependent eATP release. Moreover, JNK inhibition decreased supernatant eATP concentration and inhibited Pannexin1 activation, as determined by YoPro-1 uptake in liver cells in a dose-dependent manner. These results suggest that JNK regulates activation of Panx1 channels, and provide evidence that Pannexin1-dependent pathophysiological eATP release in lipoapoptosis is capable of stimulating migration of human monocytes, and may participate in the recruitment of monocytes in chronic liver injury induced by saturated FFA.

  3. Action of Al-ATP on the isolated working rat heart.

    Science.gov (United States)

    Korchazhkina, O; Wright, G; Exley, C

    1998-02-15

    ATP is an important extracellular messenger in the coronary vasculature of the heart. To be effective its extracellular concentration must be tightly controlled and this is achieved via ectonucleotidases located in the luminal surface of the coronary endothelium. Al-ATP is a potent inhibitor of the hydrolysis of ATP and we speculated that Al-ATP released by cells into the blood would disrupt the signalling function of extracellular ATP. We tested this hypothesis by perfusing isolated working Wistar rat hearts with buffers containing either ATP or Al-ATP. The functional parameters measured were, coronary flow, heart rate and pulsatile power. A number of control perfusions including adenosine, ATP-gamma-S and Al were used to identify those effects which might be specific to ATP and Al-ATP. Al-ATP did not appear to inhibit the function of the endothelial ectonucleotidases. Both ATP and Al-ATP produced a significant increase in coronary flow and this could be attributed to a coronary vasodilation. Interestingly, whilst the effect of ATP was reversible that of Al-ATP was not. ATP caused a reduction in heart rate which was potentiated by aluminium. The negatively chronotropic effect of Al-ATP was mediated via a mechanism which was either distinct from or in addition to the similar response known to be caused by adenosine. We have demonstrated for the first time an influence of Al-ATP on heart function. Perhaps more pertinently we present the first evidence that Al-ATP may influence the function of ATP-specific receptors.

  4. External Dentin Stimulation Induces ATP Release in Human Teeth.

    Science.gov (United States)

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain.

  5. Cellular ATP release in the lung and airway

    Directory of Open Access Journals (Sweden)

    Satoru Ito

    2016-11-01

    Full Text Available Adenosine triphosphate (ATP is a universal energy source synthesized by mitochondrial oxidative phosphorylation and cytosolic glycolysis and transported by the vesicular nucleotide transporter for storage in secretory vesicles. Extracellular ATP regulates physiological functions and homeostasis of the respiratory system and is associated with pathogenesis of respiratory diseases. Thus, modulation of ATP and purinergic signaling may be a novel therapeutic approach to pulmonary disease. ATP is released from alveolar epithelial cells, airway epithelial cells, airway smooth muscle cells, fibroblasts and endothelial cells in response to various chemical and mechanical stimuli. In addition to conductive pathways such as connexins and pannexins, vesicular exocytosis is involved in the mechanisms of ATP release from the cells. Imaging approaches enable us to visualize ATP release from not only cultured cells but also lung tissue ex vivo. Extracellular vesicles, exosomes and membrane-derived microvesicles, containing cytoplasmic proteins, mRNA and microRNA, represent important mediators of cell-to-cell communication and the intercellular microenvironment. However, it is not known whether extracellular vesicles contain ATP as an intercellular messenger. Future studies are necessary to elucidate the mechanisms of cellular ATP release and purinergic signaling in the respiratory system.

  6. Imaging Adenosine Triphosphate (ATP).

    Science.gov (United States)

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities.

  7. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K+ channels

    Science.gov (United States)

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-01-01

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K+ (KATP) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP]c) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of KATP channels in GR neurons, glucose (3 or 15 mm) caused no detectable increase in [ATP]c, monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25–luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mm) with a significant increase in [ATP]c. Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mm glucose with increases in [ATP]c. Further implicating an ATP-independent mechanism of KATP channel closure in hypothalamic neurons, removal of extracellular glucose (10 mm) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mm) of intracellular ATP. Neurons from both brain regions responded to 5 mm lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP]c. High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed KATP channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells. PMID:12381816

  8. Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.

    Directory of Open Access Journals (Sweden)

    Chia-Lin Ho

    Full Text Available Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

  9. Microglial migration mediated by ATP-induced ATP release from lysosomes

    Institute of Scientific and Technical Information of China (English)

    Ying Dou; Qing-ming Luo; Shumin Duan; Hang-jun Wu; Hui-quan Li; Song Qin; Yin-er Wang; Jing Li; Hui-fang Lou; Zhong Chen; Xiao-ming Li

    2012-01-01

    Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system.Attracted by factors released from damaged cells,microglia are recruited towards the damaged or infected site,where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris.ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury.However,the mechanisms of the long-range migration of microglia remain to be clarified.Here,we found that lysosomes in microglia contain abundant ATP and exhibit Ca2+-dependent exocytosis in response to various stimuli.By establishing an efficient in vitro chemotaxis assay,we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia,a response that was significantly inhibited in microglia treated with an agent inducing iysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice),a small GTPase required for the trafficking and exocytosis of secretory iysosomes.These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis,thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.

  10. The second extracellular loop of pore-forming subunits of ATP-binding cassette transporters for basic amino acids plays a crucial role in interaction with the cognate solute binding protein(s).

    Science.gov (United States)

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-04-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)(2) complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP(2) of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P(2) variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM.

  11. The Second Extracellular Loop of Pore-Forming Subunits of ATP-Binding Cassette Transporters for Basic Amino Acids Plays a Crucial Role in Interaction with the Cognate Solute Binding Protein(s)▿

    Science.gov (United States)

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-01-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)2 complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP2 of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P2 variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM. PMID:20154136

  12. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Jason Alan Gruenhagen

    2003-12-12

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K{sup +} and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized Cd

  13. ATP and potassium ions: a deadly combination for astrocytes

    Science.gov (United States)

    Jackson, David G.; Wang, Junjie; Keane, Robert W.; Scemes, Eliana; Dahl, Gerhard

    2014-04-01

    The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

  14. Extracellular Adenosine Triphosphate Affects Systemic and Kidney Immune Cell Populations in Pregnant Rats

    NARCIS (Netherlands)

    Spaans, Floor; Melgert, Barbro N.; Borghuis, Theo; Klok, Pieter A.; de Vos, Paul; Bakker, Winston W.; van Goor, Harry; Faas, Marijke

    PROBLEM: Changes in the systemic immune response are found in preeclampsia. This may be related to high extracellular adenosine triphosphate (ATP) levels. The question arose whether ATP could affect immune responses in pregnancy. Previously, we investigated whether ATP affected monocyte activation

  15. Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.

    Science.gov (United States)

    Kennedy, H J; Pouli, A E; Ainscow, E K; Jouaville, L S; Rizzuto, R; Rutter, G A

    1999-05-01

    Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.

  16. Hemopexin activity and extracellular ATP in the pathogenesis of preeclampsia

    NARCIS (Netherlands)

    Spaans, Floor

    2014-01-01

    Pre-eclampsie (zwangerschapsvergiftiging), de meest voorkomende zwangerschapscomplicatie, wordt gekenmerkt door hoge bloeddruk en eiwitten in de urine. We hebben geen behandeling, behalve het inleiden van de bevalling. Na de bevalling verdwijnt de aandoening. De exacte oorzaak van pre-eclampsie is n

  17. Hemopexin activity and extracellular ATP in the pathogenesis of preeclampsia

    NARCIS (Netherlands)

    Spaans, Floor

    2014-01-01

    Pre-eclampsie (zwangerschapsvergiftiging), de meest voorkomende zwangerschapscomplicatie, wordt gekenmerkt door hoge bloeddruk en eiwitten in de urine. We hebben geen behandeling, behalve het inleiden van de bevalling. Na de bevalling verdwijnt de aandoening. De exacte oorzaak van pre-eclampsie is

  18. Dual recognition unit strategy improves the specificity of the adenosine triphosphate (ATP) aptamer biosensor for cerebral ATP assay.

    Science.gov (United States)

    Yu, Ping; He, Xiulan; Zhang, Li; Mao, Lanqun

    2015-01-20

    Adenosine triphosphate (ATP) aptamer has been widely used as a recognition unit for biosensor development; however, its relatively poor specificity toward ATP against adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) essentially limits the application of the biosensors in real systems, especially in the complex cerebral system. In this study, for the first time, we demonstrate a dual recognition unit strategy (DRUS) to construct a highly selective and sensitive ATP biosensor by combining the recognition ability of aptamer toward A nucleobase and of polyimidazolium toward phosphate. The biosensors are constructed by first confining the polyimidazolium onto a gold surface by surface-initiated atom transfer radical polymerization (SI-ATRP), and then the aptamer onto electrode surface by electrostatic self-assembly to form dual-recognition-unit-functionalized electrodes. The constructed biosensor based on DRUS not only shows an ultrahigh sensitivity toward ATP with a detection limit down to the subattomole level but also an ultrahigh selectivity toward ATP without interference from ADP and AMP. The constructed biosensor is used for selective and sensitive sensing of the extracellular ATP in the cerebral system by combining in vivo microdialysis and can be used as a promising neurotechnology to probing cerebral ATP concentration.

  19. KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

    Directory of Open Access Journals (Sweden)

    Javier Pizarro-Delgado

    Full Text Available Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1. ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering and second (amplifying phases of glucose-induced insulin secretion.

  20. Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts

    Science.gov (United States)

    Shiozaki, Yuta; Sato, Masaki; Kimura, Maki; Sato, Toru; Tazaki, Masakazu; Shibukawa, Yoshiyuki

    2017-01-01

    ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1–P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury. PMID:28163685

  1. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

    OpenAIRE

    Rak, Malgorzata; Tzagoloff, Alexander

    2009-01-01

    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  2. Real-time imaging of inflation-induced ATP release in the ex vivo rat lung.

    Science.gov (United States)

    Furuya, Kishio; Tan, Ju Jing; Boudreault, Francis; Sokabe, Masahiro; Berthiaume, Yves; Grygorczyk, Ryszard

    2016-11-01

    Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into air spaces, brief inflation of such edematous lung (1 s, ∼20 cmH2O) induced transient (lungs and provides the first direct evidence of inflation-induced ATP release in lung air spaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function. Copyright © 2016 the American Physiological Society.

  3. 异丙酚在不同pH值环境下对苯甲酰苯甲酸ATP诱发的巨噬细胞嘌呤2X7受体电流的影响%Effect of propofol on BzATP induced P2X7-gated currents in RAW264.7 macrophages under different extracellular pH values

    Institute of Scientific and Technical Information of China (English)

    刘红亮; 戴体俊

    2011-01-01

    To investigate the sensitivity of P2X7 receptor to its specific agonist BZATP and the effect of propofol on P2X7-gated currents in RAW264. 7 macrophages under different extracellular pH values. METHODS-RAW264. 7 cells were cultured, and whole-cell patch clamp technique was used. BZATP (10 - 10000 pimol/L) was applied for 5 seconds, the currents were recorded, and the EC50 value of BZATP was achieved under the extracellular pH value of 7. 4 or 6. 0. Propofol (1 -100 jumol/L) was applied to the cells for 1 min, then BZATP with two times EC50 value was applied, the IC50 or EC50 value of propofol was achieved under different extracellular pH values. To investigate the effect of propofol on the dose-response curve of B2ATP under different extracellular pH values, propofol with IC50 value was applied, and BZATP (10-10000 fimol/L) was applied for 5 seconds, the EC50 value of BZATP was achieved. RESULTS :BZATP could induce the inward cur-rents in a dose-dependent manner, and the EC50 value of BZATP was (112 ±26) μmol/L and (643 ±87) μmol/L at extracellular pH value of 7. 4 or 6. 0. Propofol could inhibit P2X7-gated currents at pH 7. 4, and the IC50 value was (31 ±6) μmol/L; but propofol increased P2X7-ga-ted currents at pH 6. 0, and the EC50 value was (38 ±6) fxmol/L. The IC50 value of propofol made the dose-response curve of BZATP shifted rightward at pH 7.4, or leftward at pH 6.0. CONCLUSION: The sensitivity of P2X7 receptor to BZATP decreases when extracellular pH value changes from 7. 4 to 6. 0. Propofol with clinically related concentrations could inhibit P2X7-ga-ted currents at pH 7. 4, and increase P2X7-gated currents at pH 6. 0 extracellularly.%目的:观察在不同pH值条件下巨噬细胞膜嘌呤2X7(P2X7)受体电流对其特异性激动剂苯甲酰苯甲酸ATP (BZATP)的敏感性及异丙酚对P2X7受体电流的影响.方法:应用培养的RAW264.7巨噬细胞,采用全细胞膜片钳技术,向细胞施加BZ ATP(10~10000 μmol/L)5 s,记录电流,

  4. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  5. ATP Is Required and Advances Cytokine-Induced Gap Junction Formation in Microglia In Vitro

    Directory of Open Access Journals (Sweden)

    Pablo J. Sáez

    2013-01-01

    Full Text Available Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs and gap junction channels (GJCs, affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-α/IFN-γ-induced dye coupling, probably through the induction of IL-1β release. Moreover, TNF-α/IFN-γ, but not TNF-α plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-α/IFN-γ, but not TNF-α plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-α/IFN-γ in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.

  6. Interdependence of ATP signalling and pannexin channels; the servant was really the master all along?

    Science.gov (United States)

    Jackson, Michael F

    2015-12-15

    Pannexin channels are recognized as important conduits for the release of ATP, which contributes to purinergic signalling. Pathologically, ATP release via these channels acts as a find-me signal for apoptotic cell clearance. Accordingly, there is considerable and growing interest in understanding the function and regulation of pannexin channels. In a recent issue of the Biochemical Journal, Boyce et al. provide evidence that the surface expression of pannexin channels is regulated by extracellular ATP. They propose a model in which ATP triggers pannexin channel internalization through a pathway involving clathrin- and caveolin-independent entry into early endosomes. Intriguingly, their evidence suggests that internalization is initiated through the association of ATP with pannexin channels themselves as well as ionotropic purinergic receptor 7 (P2X7) receptors.

  7. Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy

    DEFF Research Database (Denmark)

    Sørensen, Christiane Elisabeth; Novak, Ivana

    2001-01-01

    The energy providing substrate ATP can be released from various cells and act extracellularly to regulate the same cells or neighboring cells. However, the pathway for ATP release and the eliciting physiological stimulus are unclear. Recently, we showed that ATP activates P2X and P2Y purinergic...... overlapping with those marked by acridine orange and LysoTracker Red. In functional studies we show that native pancreatic acini release ATP in response to various stimuli but most importantly to cholinergic stimulation, a very likely physiological stimulus in this epithelium. In a close vicinity of acini we...

  8. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    Energy Technology Data Exchange (ETDEWEB)

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  9. Adenosine triphosphate (ATP) reduces amyloid-β protein misfolding in vitro.

    Science.gov (United States)

    Coskuner, Orkid; Murray, Ian V J

    2014-01-01

    Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-β protein (Aβ) levels. Since levels of ATP decrease over the course of the disease and Aβ is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both Aβ (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aβ mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant Aβ42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aβ fibrils in solution. Experimentally, both ATP and ADP reduced Aβ misfolding at physiological intracellular concentrations, with thresholds at ~500 μM and 1 mM respectively. This inhibition of Aβ misfolding is specific; requiring Tyr10 of Aβ and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with Aβ and reduction of Aβ misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular Aβ mirrors lowered extracellular ATP levels. Last, the findings suggest that Aβ conformation change may be a sensor of metabolic dysfunction.

  10. Molecular mechanism for H(2)S-induced activation of K(ATP) channels.

    Science.gov (United States)

    Jiang, Bo; Tang, Guanghua; Cao, Kun; Wu, Lingyun; Wang, Rui

    2010-05-15

    Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.

  11. ATP as a biomarker of viable microorganisms in clean-room facilities

    Science.gov (United States)

    Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T.; Kern, Roger

    2003-01-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

  12. ATP as a biomarker of viable microorganisms in clean-room facilities

    Science.gov (United States)

    Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T.; Kern, Roger

    2003-01-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

  13. Substantia nigra osmoregulation: taurine and ATP involvement.

    Science.gov (United States)

    Morales, Ingrid; Dopico, Jose G; Sabate, Magdalena; Gonzalez-Hernandez, Tomas; Rodriguez, Manuel

    2007-05-01

    An extracellular nonsynaptic taurine pool of glial origin was recently reported in the substantia nigra (SN). There is previous evidence showing taurine as an inhibitory neurotransmitter in the SN, but the physiological role of this nonsynaptic pool of taurine has not been explored. By using microdialysis methods, we studied the action of local osmolarity on the nonsynaptic taurine pool in the SN of the rat. Hypoosmolar pulses (285-80 mosM) administered in the SN by the microdialysis probe increased extrasynaptic taurine in a dose-dependent way, a response that was counteracted by compensating osmolarity with choline. The opposite effect (taurine decrease) was observed when osmolarity was increased. Under basal conditions, the blockade of either the AMPA-kainate glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dionine disodium or the purinergic receptors with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid modified the taurine concentration, suggesting that both receptors modulate the extrasynaptic pool of taurine. In addition, these drugs decreased the taurine response to hypoosmolar pulses, suggesting roles for glutamatergic and purinergic receptors in the taurine response to osmolarity. The participation of purinergic receptors was also supported by the fact that ATP (which, under basal conditions, increased the extrasynaptic taurine in a dose-dependent way) administered in doses saturating purinergic receptors also decreased the taurine response to hypoosmolarity. Taken together, present data suggest osmoregulation as a role of the nonsynaptic taurine pool of the SN, a function that also involves glutamate and ATP and that could influence the nigral cell vulnerability in Parkinson's disease.

  14. Function and expression study uncovered hepatocyte plasma membrane ecto-ATP synthase as a novel player in liver regeneration.

    Science.gov (United States)

    Taurino, Federica; Giannoccaro, Caterina; Sardanelli, Anna Maria; Cavallo, Alessandro; De Luca, Elisa; Santacroce, Salvatore; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2016-08-15

    ATP synthase, canonically mitochondrially located, is reported to be ectopically expressed on the plasma membrane outer face of several cell types. We analysed, for the first time, the expression and catalytic activities of the ecto- and mitochondrial ATP synthase during liver regeneration. Liver regeneration was induced in rats by two-thirds partial hepatectomy. The protein level and the ATP synthase and/or hydrolase activities of the hepatocyte ecto- and mitochondrial ATP synthase were analysed on freshly isolated hepatocytes and mitochondria from control, sham-operated and partial hepatectomized rats. During the priming phase of liver regeneration, 3 h after partial hepatectomy, liver mitochondria showed a marked lowering of the ATP synthase protein level that was reflected in the impairment of both ATP synthesis and hydrolysis. The ecto-ATP synthase level, in 3 h partial hepatectomized hepatocytes, was decreased similarly to the level of the mitochondrial ATP synthase, associated with a lowering of the ecto-ATP hydrolase activity coupled to proton influx. Noteworthily, the ecto-ATP synthase activity coupled to proton efflux was completely inhibited in 3 h partial hepatectomized hepatocytes, even in the presence of a marked intracellular acidification that would sustain it as in control and sham-operated hepatocytes. At the end of the liver regeneration, 7 days after partial hepatectomy, the level and the catalytic activities of the ecto- and mitochondrial ATP synthase reached the control and sham-operated values. The specific modulation of hepatocyte ecto-ATP synthase catalytic activities during liver regeneration priming phase may modulate the extracellular ADP/ATP levels and/or proton influx/efflux trafficking, making hepatocyte ecto-ATP synthase a candidate for a novel player in the liver regeneration process. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  15. The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

    OpenAIRE

    Suzuki, Haruka; Kuroda, Hiroshi; Yukawa, Yasushi; Sugiura, Masahiro

    2011-01-01

    The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the ...

  16. Stimulation-dependent release, breakdown, and action of endogenous ATP in mouse hemidiaphragm preparation: the possible role of ATP in neuromuscular transmission.

    Science.gov (United States)

    Vizi, E S; Nitahara, K; Sato, K; Sperlágh, B

    2000-07-01

    In this study the in vitro mouse phrenic nerve- hemidiaphragm preparation was utilized to study the release and extracellular catabolism of endogenous ATP and its action on the postsynaptic site, i.e. on the contraction force evoked by nerve stimulation. ATP, measured by the luciferin-luciferase assay, was released stimulation-dependently from the mouse hemidiaphragm in response to electrical field stimulation at 10 Hz. Blockade of the Na(+) channel activity by tetrodotoxin inhibited the majority of the release of ATP in response to stimulation, showing that it is related to neuronal activity. The nicotinic receptor antagonists d-tubocurarine, and alpha-bungarotoxin and cooling the bath temperature to 7 degrees C also reduced stimulation-induced ATP outflow, suggesting that nicotinic receptors are responsible for the part of the release of ATP that is released from postsynaptic sites in a carrier-mediated manner. Exogenous ATP (20-500 microM) added to the bath was degraded to ADP and AMP by the action of ectoATPase and ectoATPdiphosphohydrolase; the K(m) and v(max) values of these enzymes were 185.8 microM and 55.16 nmol/min.g respectively. However, the total amount of nucleotides ([ATP+ADP+AMP]) was increased after the addition of ATP, indicating that ATP itself promoted further adenine nucleotide release. Twitch contractions of the rat hemidiaphragm preparation evoked by low frequency electrical stimulation was blocked concentration-dependently by the non-depolarizing muscle relaxants d-tubocurarine and pancuronium. Suramin (100 microM-1 mM) reversed neuromuscular blockade by d-tubocurarine and pancuronium; i.e., it shifted their concentration-response curves to the right Taken together our data, that endogenous ATP is released by stimulation and subsequently catabolized in the hemidiaphragm preparation and that suramin inhibits ecto-ATPase activity could be interpreted as meaning that suramin prolongs the action of endogenous ATP to elicit twitch contraction

  17. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects.

    Science.gov (United States)

    Kojima, Shuji; Ohshima, Yasuhiro; Nakatsukasa, Hiroko; Tsukimoto, Mitsutoshi

    2017-01-01

    Adenosine triphosphate (ATP) serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy) induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells.

  18. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects

    Directory of Open Access Journals (Sweden)

    Shuji Kojima

    2017-02-01

    Full Text Available Adenosine triphosphate (ATP serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells.

  19. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects

    Science.gov (United States)

    Ohshima, Yasuhiro; Nakatsukasa, Hiroko; Tsukimoto, Mitsutoshi

    2017-01-01

    Adenosine triphosphate (ATP) serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy) induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells.

  20. Real-time imaging of ATP release induced by mechanical stretch in human airway smooth muscle cells.

    Science.gov (United States)

    Takahara, Norihiro; Ito, Satoru; Furuya, Kishio; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-12-01

    Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca(2+)-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

  1. Involvement of connexin43 hemichannel in ATP release after γ-irradiation

    Science.gov (United States)

    Ohshima, Yasuhiro; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Kojima, Shuji

    2012-01-01

    Ionizing radiation induces biological effects not only in irradiated cells but also in non-irradiated cells, which is called the bystander effect. Recently, in vivo and in vitro experiments have suggested that both gap junction hemichannel connexin43 (Cx43) and extracellular adenosine triphosphate (ATP) released from cells play a role in the bystander effect. We have reported that γ-irradiation induces ATP release from B16 melanoma cells, which is dependent on the P2X7 receptor. However, the mechanism of ATP release caused by irradiation remains unclear. We here show the involvement of Cx43 in P2X7 receptor-dependent ATP release after 0.5 Gy γ-irradiation. Inhibitors of gap junction hemichannels and an inhibitory peptide for Cx43 (gap26), but not an inhibitory peptide for pannexin1 (Panx1), significantly blocked γ-irradiation-induced ATP release from B16 melanoma cells. We confirmed high expression of Cx43 mRNA in B16 melanoma cells. These results suggest involvement of Cx43 in radiation-induced ATP release. We found that after 0.5 Gy γ-irradiation tyrosine phosphorylation was significantly blocked by P2X7 receptor antagonist, but not gap26, suggesting that tyrosine phosphorylation is a downstream event from the P2X7 receptor. Since tyrosine kinase inhibitor significantly suppressed radiation-induced ATP release, tyrosine phosphorylation appears to play an important role in the Cx43-mediated ATP release downstream of the P2X7 receptor. In conclusion, the Cx43 hemichannel, which lies downstream of the P2X7 receptor, is involved in ATP release in response to radiation. Our results suggest a novel mechanism for radiation-induced biological effects mediated by both ATP and Cx43. PMID:22843620

  2. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  3. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  4. ATP Release and Effects in Pancreas

    DEFF Research Database (Denmark)

    Novak, Ivana; Amstrup, Jan; Henriksen, Katrine Lütken

    2003-01-01

    ATP and other nucleotides are released from various cells, but the pathway and physiological stimulus for ATP release are often unclear. The focus of our studies is the understanding of ATP release and signaling in rat exocrine pancreas. In acinar suspension mechanical stimulation, hypotonic shock...... and, most importantly, cholinergic stimulation released 5-20nM ATP into the medium, as monitored by luminescence of the luciferin/luciferase reaction. ATP release was visualized at the single acinus level as luciferin consumption detected by confocal laser scanning microscopy (CLSM). The estimated ATP...... concentrations were higher, about 10µM, around acinar cells after cholinergic stimulation. Fluorescence of quinacrine and MANT-ATP indicated that some ATP is stored in secretory granules. Pancreatic acini have transcripts for P2X1, P2X4, P2Y2, and P2Y4 receptors, but measurements of Ca2+ signals in isolated...

  5. A secreted tyrosine kinase acts in the extracellular environment.

    Science.gov (United States)

    Bordoli, Mattia R; Yum, Jina; Breitkopf, Susanne B; Thon, Jonathan N; Italiano, Joseph E; Xiao, Junyu; Worby, Carolyn; Wong, Swee-Kee; Lin, Grace; Edenius, Maja; Keller, Tracy L; Asara, John M; Dixon, Jack E; Yeo, Chang-Yeol; Whitman, Malcolm

    2014-08-28

    Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.

  6. ATPase Activity Measurements Using Radiolabeled ATP

    NARCIS (Netherlands)

    Swarts, H.G.; Koenderink, J.B.

    2016-01-01

    ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [gamma-(32)P]ATP, which is hydrolyzed by P-type ATPases

  7. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured...

  8. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz;

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured...

  9. 2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

    Science.gov (United States)

    Schachter, Julieta; Valadao, Ana Luiza Chaves; Aguiar, Renato Santana; Barreto-de-Souza, Victor; Rossi, Atila Duque; Arantes, Pablo Ricardo; Verli, Hugo; Quintana, Paula Gabriela; Heise, Norton; Tanuri, Amilcar; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis

    2014-01-01

    The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

  10. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

    DEFF Research Database (Denmark)

    Ostergaard, L; Teilum, K; Mirza, O;

    2000-01-01

    to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover......Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown......-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant...

  11. Effect of ATP on intracellular pH in pancreatic ducts involves P2X7 receptors

    DEFF Research Database (Denmark)

    Henriksen, Katerine L; Novak, Ivana

    2003-01-01

    Pancreatic acini release ATP, which can stimulate HCO3--secreting ducts that express purinergic receptors from both P2X and P2Y families. The aim of this study was to investigate whether extracellular ATP affects HCO3- or H+ transport across the plasma membrane of intralobular ducts, and determine......, experiments were performed in solutions with or without bicarbonate buffers (+BIC or -BIC), with amiloride derivative EIPA, or with low extracellular Cl- concentrations. Although these transporters contributed to pHi recovery from acidosis, ATP had no effect. Nevertheless, ATP induced a small and reversible...... decrease in pHi by 0.07+/-0.02 pH-units and BzATP decreased pHi by 0.29+/-0.07 pH-units in -BIC (n=10, 11). These effects were abolished by Brilliant Blue and in Ca2+-free solutions. Our study shows that the pHi effect of ATP is mediated by P2X7 receptors. However, ATP does not affect H+/HCO3- transporters...

  12. Antiphospholipid Antibodies Bind ATP: A putative Mechanism for the Pathogenesis of Neuronal Dysfunction

    Directory of Open Access Journals (Sweden)

    J. Chapman

    2005-01-01

    Full Text Available Antiphospholipid antibodies (aPL generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA. Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS. IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (>50% inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound β2-glycoprotein-I (β2-GPI, also known as apolipoprotein H suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of β2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of β2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through β2-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters.

  13. ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

    Science.gov (United States)

    Song, Shanshan; Jacobson, Krista N; McDermott, Kimberly M; Reddy, Sekhar P; Cress, Anne E; Tang, Haiyang; Dudek, Steven M; Black, Stephen M; Garcia, Joe G N; Makino, Ayako; Yuan, Jason X-J

    2016-01-15

    Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. Copyright

  14. Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

    Science.gov (United States)

    Bertrand, C A; Danahay, H; Poll, C T; Laboisse, C; Hopfer, U; Bridges, R J

    2004-02-01

    ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

  15. Extracellular calcium sensing and extracellular calcium signaling

    Science.gov (United States)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  16. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution......, since the method is very sensitive (detects 0.5 ng ATP/L) and results are obtained within minutes. When calculating the ATP value a number of parameters need to be considered. These were investigate by use of two different reagent kits (PCP-kit and Lumin(ATE)/Lumin(EX)-kit), internal standard...... and an Advance Coupe luminometer. The investigations showed a 60 times higher response of the PCP-kit, making it more suitable for measurement of samples with low ATP content. ATP-standard dilutions prepared in tap water were stable for at least 15 months when stored frozen at -80ºC, and storage of large...

  17. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution......, since the method is very sensitive (detects 0.5 ng ATP/L) and results are obtained within minutes. When calculating the ATP value a number of parameters need to be considered. These were investigate by use of two different reagent kits (PCP-kit and Lumin(ATE)/Lumin(EX)-kit), internal standard...... and an Advance Coupe luminometer. The investigations showed a 60 times higher response of the PCP-kit, making it more suitable for measurement of samples with low ATP content. ATP-standard dilutions prepared in tap water were stable for at least 15 months when stored frozen at -80ºC, and storage of large...

  18. Glibenclamide decreases ATP-induced intracellular calcium transient elevation via inhibiting reactive oxygen species and mitochondrial activity in macrophages.

    Directory of Open Access Journals (Sweden)

    Duo-ling Li

    Full Text Available Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP-induced intracellular calcium ([Ca(2+]i handling in Raw 264.7 macrophages. In the present study, [Ca(2+]i transient, reactive oxygen species (ROS and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+ channel blockers had no effect on the resting [Ca(2+]i of Raw 264.7 cells. Extracellular ATP (100 µM induced [Ca(2+]i transient elevation independent of extracellular Ca(2+. The transient elevation was inhibited by an ROS scavenger (tiron and mitochondria inhibitor (rotenone. Glibenclamide and 5-hydroxydecanoate (5-HD also decreased ATP-induced [Ca(2+]i transient elevation, but pinacidil and other unselective K(+ channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+]i transient induced by extracellular thapsigargin (Tg, 1 µM. Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

  19. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun [Iowa State Univ., Ames, IA (United States)

    2008-12-18

    mainly used a fluorescence method; CL detection is limited because of the difficulty to introduce enough D-luciferin molecules. Since dehydration could easily cause proper size holes in bacterial cell membranes and facilitate D-luciferin diffusion, we used this method and recorded CL from individual cells each hour after induction. The CL light intensity from each individual cell was integrated and gene expression levels of two strain types were compared. Based on our calculation, the overall sensitivity of our system is already approaching the single enzyme level. The median enzyme number inside a single bacterium from the higher expression strain after 2 hours induction was quantified to be about 550 molecules. Finally we imaged ATP release from astrocyte cells. Upon mechanical stimulation, astrocyte cells respond by increasing intracellular Ca 2+ level and releasing ATP to extracellular spaces as signaling molecules. The ATP release imaged by direct CL imaging using free firefly luciferase and D-luciferin outside cells reflects the transient release as well as rapid ATP diffusion. Therefore ATP release detection at the cell surface is critical to study the ATP release mechanism and signaling propagation pathway. We realized this cell surface localized ATP release imaging detection by immobilizing firefly luciferase to streptavidin beads that attached to the cell surface via streptavidin-biotin interactions. Both intracellular Ca2+ propagation wave and extracellular ATP propagation wave at the cell surface were recorded with fluorescence and CL respectively. The results imply that at close distances from the stimulation center (<120 μm) extracellular ATP pathway is faster, while at long distances (>120 μm) intracellular Ca2+ signaling through gap junctions seems more effective.

  20. The ATP-P2X7 signalling axis is dispensable for obesity-associated inflammasome activation in adipose tissue

    NARCIS (Netherlands)

    Sun, S.; Xia, S.; Ji, Y.; Kersten, A.H.; Qi, L.

    2012-01-01

    Inflammasome activation in adipose tissue has been implicated in obesity-associated insulin resistance and type 2 diabetes. However, when and how inflammasome is activated in adipose tissue remains speculative. Here we test the hypothesis that extracellular ATP, a potent stimulus of inflammasome in

  1. The ATP-P2X7 signalling axis is dispensable for obesity-associated inflammasome activation in adipose tissue

    NARCIS (Netherlands)

    Sun, S.; Xia, S.; Ji, Y.; Kersten, A.H.; Qi, L.

    2012-01-01

    Inflammasome activation in adipose tissue has been implicated in obesity-associated insulin resistance and type 2 diabetes. However, when and how inflammasome is activated in adipose tissue remains speculative. Here we test the hypothesis that extracellular ATP, a potent stimulus of inflammasome in

  2. ATP as a peripheral mediator of pain.

    Science.gov (United States)

    Hamilton, S G; McMahon, S B

    2000-07-01

    This article reviews the extent to which recent studies substantiate the hypothesis that ATP functions as a peripheral pain mediator. The discovery of the P2X family of ion channels (for which ATP is a ligand) and, in particular, the highly selective distribution of the P2X(3) receptor within the rat nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. ATP elicits excitatory inward currents in small diameter sensory ganglion cells. These currents resemble those elicited by ATP on recombinantly expressed heteromeric P2X(2/3) channels as well as homomultimers consisting of P2X(2) and P2X(3). In vivo behavioural models have characterised the algogenic properties of ATP in normal conditions and in models of peripheral sensitisation. In humans, iontophoresis of ATP induces modest pain. In rats and humans the response is dependent on capsaicin sensitive neurons and is augmented in the presence of inflammatory mediators. Since ATP can be released in the vicinity of peripheral nociceptive terminals under a variety of conditions, there exists a purinergic chain of biological processes linking tissue damage to pain perception. The challenge remains to prove a physiological role for endogenous ATP in activating this chain of events.

  3. Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP.

    Science.gov (United States)

    Karmakar, Mausita; Katsnelson, Michael A; Dubyak, George R; Pearlman, Eric

    2016-02-15

    Although extracellular ATP is abundant at sites of inflammation, its role in activating inflammasome signalling in neutrophils is not well characterized. In the current study, we demonstrate that human and murine neutrophils express functional cell-surface P2X7R, which leads to ATP-induced loss of intracellular K(+), NLRP3 inflammasome activation and IL-1β secretion. ATP-induced P2X7R activation caused a sustained increase in intracellular [Ca(2+)], which is indicative of P2X7R channel opening. Although there are multiple polymorphic variants of P2X7R, we found that neutrophils from multiple donors express P2X7R, but with differential efficacies in ATP-induced increase in cytosolic [Ca(2+)]. Neutrophils were also the predominant P2X7R-expressing cells during Streptococcus pneumoniae corneal infection, and P2X7R was required for bacterial clearance. Given the ubiquitous presence of neutrophils and extracellular ATP in multiple inflammatory conditions, ATP-induced P2X7R activation and IL-1β secretion by neutrophils likely has a significant, wide ranging clinical impact.

  4. Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells.

    Science.gov (United States)

    Toma, Ildikó; Bansal, Eric; Meer, Elliott J; Kang, Jung Julie; Vargas, Sarah L; Peti-Peterdi, János

    2008-06-01

    Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.

  5. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

    Science.gov (United States)

    Xing, Shu; Grol, Matthew W; Grutter, Peter H; Dixon, S Jeffrey; Komarova, Svetlana V

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca(2+)]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca(2+)]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K 1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

  6. (31)P-MRS of healthy human brain: ATP synthesis, metabolite concentrations, pH, and T1 relaxation times.

    Science.gov (United States)

    Ren, Jimin; Sherry, A Dean; Malloy, Craig R

    2015-11-01

    The conventional method for measuring brain ATP synthesis is (31)P saturation transfer (ST), a technique typically dependent on prolonged pre-saturation with γ-ATP. In this study, ATP synthesis rate in resting human brain is evaluated using EBIT (exchange kinetics by band inversion transfer), a technique based on slow recovery of γ-ATP magnetization in the absence of B1 field following co-inversion of PCr and ATP resonances with a short adiabatic pulse. The unidirectional rate constant for the Pi → γ-ATP reaction is 0.21 ± 0.04 s(-1) and the ATP synthesis rate is 9.9 ± 2.1 mmol min(-1)  kg(-1) in human brain (n = 12 subjects), consistent with the results by ST. Therefore, EBIT could be a useful alternative to ST in studying brain energy metabolism in normal physiology and under pathological conditions. In addition to ATP synthesis, all detectable (31)P signals are analyzed to determine the brain concentration of phosphorus metabolites, including UDPG at around 10 ppm, a previously reported resonance in liver tissues and now confirmed in human brain. Inversion recovery measurements indicate that UDPG, like its diphosphate analogue NAD, has apparent T1 shorter than that of monophosphates (Pi, PMEs, and PDEs) but longer than that of triphosphate ATP, highlighting the significance of the (31)P-(31)P dipolar mechanism in T1 relaxation of polyphosphates. Another interesting finding is the observation of approximately 40% shorter T1 for intracellular Pi relative to extracellular Pi, attributed to the modulation by the intracellular phosphoryl exchange reaction Pi ↔ γ-ATP. The sufficiently separated intra- and extracellular Pi signals also permit the distinction of pH between intra- and extracellular environments (pH 7.0 versus pH 7.4). In summary, quantitative (31)P MRS in combination with ATP synthesis, pH, and T1 relaxation measurements may offer a promising tool to detect biochemical alterations at early stages of brain dysfunctions and diseases.

  7. Glioactive ATP controls BDNF recycling in cortical astrocytes

    Science.gov (United States)

    Vignoli, Beatrice; Canossa, Marco

    2017-01-01

    ABSTRACT We have recently reported that long-term memory retention requires synaptic glia for proBDNF uptake and recycling. Through the recycling course, glial cells release endocytic BDNF, a mechanism that is activated in response to glutamate via AMPA and mGluRI/II receptors. Cortical astrocytes express receptors for many different transmitters suggesting for a complex signaling controlling endocytic BDNF secretion. Here, we demonstrated that the extracellular nucleotide ATP, activating P2X and P2Y receptors, regulates endocytic BDNF secretion in cultured astrocytes. Our data indicate that distinct glioactive molecules can participate in BDNF glial recycling and suggest that cortical astrocytes contributing to neuronal plasticity can be influenced by neurotransmitters in tune with synaptic needs.

  8. Extracellular Gd-CA

    DEFF Research Database (Denmark)

    Thomsen, Henrik S; Marckmann, Peter

    2008-01-01

    Until recently it was believed that extracellular gadolinium-based contrast agents were safe for both the kidneys and all other organs within the dose range up to 0.3 mmol/kg body weight. However, in 2006, it was demonstrated that some gadolinium-based contrast agents may trig the development of ...

  9. No effect of aluminium upon the hydrolysis of ATP in the coronary circulation of the isolated working rat heart.

    Science.gov (United States)

    Korchazhkina, O; Wright, G; Exley, C

    1999-08-30

    Adenosine 5'-triphosphate (ATP) is now recognised as an important extracellular signalling molecule. Its action at a number of specific receptors is mediated by the activity of ectonucleotidases. We have optimised a high performance liquid chromatography (HPLC) method to allow the simultaneous determination of ATP, and the products of its hydrolysis, in the coronary effluent of an isolated working rat heart. The method is extremely sensitive allowing picomolar quantities of product to be determined. We have used this method to investigate the influence of aluminium on the hydrolysis of ATP by an ecto-ATPase located in the luminal surface of the coronary endothelium of the rat heart. Aluminium did not influence the hydrolysis of ATP by this enzyme.

  10. ATP and Presentation Service for Mizar Formalizations

    CERN Document Server

    Urban, Josef; Sitcliffe, Geoff

    2011-01-01

    This paper describes the Automated Reasoning for Mizar (MizAR) service, which integrates several automated reasoning, artificial intelligence, and presentation tools with Mizar and its authoring environment. The service provides ATP assistance to Mizar authors in finding and explaining proofs, and offers generation of Mizar problems as challenges to ATP systems. The service is based on a sound translation from the Mizar language to that of first-order ATP systems, and relies on the recent progress in application of ATP systems in large theories containing tens of thousands of available facts. We present the main features of MizAR services, followed by an account of initial experiments in finding proofs with the ATP assistance. Our initial experience indicates that the tool offers substantial help in exploring the Mizar library and in preparing new Mizar articles.

  11. [ATP in the metabolism of ruminants].

    Science.gov (United States)

    Bergner, H

    1991-10-01

    The ATP yield from the carbohydrates of anaerobically living microorganisms in the rumen amounts to only 5-10% of the ATP yield of the intermediary metabolism in the presence of oxygen. Vital functions and thus microbial protein synthesis are due to protein degradation in the rumen. The ATP yield in the intermediary metabolism of ruminants is mainly achieved from propionate and microbial protein by means of gluconeogenesis because the absorption of glucose from digested starch is very low. The relationships between ATP yield in the rumen and the processes of glucose provision for the production of lactose as well as the protein content of the milk are shown. As important processes of ATP production in microorganisms from easily soluble carbohydrates take place in silage preparations before feed intake, the corresponding consequences for the metabolism of high-performance cows fed with silage are shown.

  12. Thermodynamics of proton transport coupled ATP synthesis.

    Science.gov (United States)

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase.

  13. RNAi-mediated silencing of Atp6i and Atp6i haploinsufficiency prevents both bone loss and inflammation in a mouse model of periodontal disease.

    Directory of Open Access Journals (Sweden)

    Hongbing Jiang

    Full Text Available Periodontal disease affects about 80% of adults in America, and is characterized by oral bacterial infection-induced gingival inflammation, oral bone resorption, and tooth loss. Periodontitis is also associated with other diseases such as rheumatoid arthritis, diabetes, and heart disease. Although many efforts have been made to develop effective therapies for this disease, none have been very effective and there is still an urgent need for better treatments and preventative strategies. Herein we explored for the first time the possibility that adeno-associated virus (AAV-mediated RNAi knockdown could be used to treat periodontal disease with improved efficacy. For this purpose, we used AAV-mediated RNAi knockdown of Atp6i/TIRC7 gene expression to target bone resorption and gingival inflammation simultaneously. Mice were infected with the oral pathogen Porphyromonas gingivalis W50 (P. gingivalis in the maxillary periodontium to induce periodontitis. We found that Atp6i depletion impaired extracellular acidification and osteoclast-mediated bone resorption. Furthermore, local injection of AAV-shRNA-Atp6i/TIRC7 into the periodontal tissues in vivo protected mice from P. gingivalis infection-stimulated bone resorption by >85% and decreased the T-cell number in periodontal tissues. Notably, AAV-mediated Atp6i/TIRC7 knockdown also reduced the expression of osteoclast marker genes and inflammation-induced cytokine genes. Atp6i(+/- mice with haploinsufficiency were similarly protected from P. gingivalis infection-stimulated bone loss and gingival inflammation. This suggests that AAV-shRNA-Atp6i/TIRC7 therapeutic treatment may significantly improve the health of millions who suffer from P. gingivalis-mediated periodontal disease.

  14. Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors.

    Science.gov (United States)

    Xia, Jingsheng; Lim, Jason C; Lu, Wennan; Beckel, Jonathan M; Macarak, Edward J; Laties, Alan M; Mitchell, Claire H

    2012-05-15

    Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide (10)panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X(7) receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X(7) receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X(7) receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X(7) receptor, is neuroprotective for stretched neurons.

  15. Feasibility study of B16 melanoma therapy using oxidized ATP to target purinergic receptor P2X7.

    Science.gov (United States)

    Hattori, Fumie; Ohshima, Yasuhiro; Seki, Shizuka; Tsukimoto, Mitsutoshi; Sato, Mitsuru; Takenouchi, Takato; Suzuki, Akina; Takai, Erina; Kitani, Hiroshi; Harada, Hitoshi; Kojima, Shuji

    2012-11-15

    The P2X7 receptor is not only involved in cell proliferation, but also acts as an adenosine 5'-triphosphate (ATP)-gated non-selective channel, and its expression is increased in human melanoma. An irreversible antagonist of P2X7, such as oxidized ATP (oxATP), might block P2X7 receptor-mediated ATP release and proliferative signaling. Therefore, we carried out basic studies to test this idea and to examine the feasibility of using oxATP to treat B16 melanoma. We first found that low-pH conditions (mimicking the hypoxia and acidosis commonly seen in solid tumors) induced P2X7 receptor-mediated ATP release from B16 melanoma cells. Then, we compared the proliferation rates of B16 melanoma wild-type cells and B16 P2X7 receptor-knockdown clone (P2X7-KDC) cells in the presence of P2X7 agonists. The proliferation rate, as well as the ATP release, of agonist-treated P2X7-KDC cells was lower than that of agonist-treated wild-type cells. Next, the effect of P2X7 antagonist oxATP on B16 melanoma cell growth was examined in vitro and in vivo. oxATP significantly decreased B16 melanoma cell proliferation in vitro, and also significantly inhibited tumor growth in B16 melanoma-bearing mice. These data indicate that extracellularly released ATP may serve as an intercellular signaling molecule. We propose that the P2X7 receptor is a promising target for treatment of solid tumors.

  16. Inseparable tandem: evolution chooses ATP and Ca2+ to control life, death and cellular signalling.

    Science.gov (United States)

    Plattner, Helmut; Verkhratsky, Alexei

    2016-08-05

    From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca(2+) to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca(2+) as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca(2+) as a universal signalling ion; similarly, Ca(2+) is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca(2+) started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca(2+) interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca(2+) low forced cells to restrict Ca(2+) signals in space and time and to develop energetically favourable Ca(2+) signalling and Ca(2+) microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca(2+)-regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca(2+) signalling. Similar to atmospheric oxygen, Ca(2+) must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca(2+) homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca(2+) and ATP have been exploited in evolution, thus turning an originally

  17. Receptor-like activity evoked by extracellular ADP in Arabidopsis root epidermal plasma membrane.

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M

    2011-07-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca(2+) influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca(2+) in root epidermal protoplasts. An inward Ca(2+)-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca(2+) efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

  18. Receptor-Like Activity Evoked by Extracellular ADP in Arabidopsis Root Epidermal Plasma Membrane1

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M.

    2011-01-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca2+ influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca2+ in root epidermal protoplasts. An inward Ca2+-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca2+ efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM. PMID:21562328

  19. The two-hit hypothesis for neuroinflammation: role of exogenous ATP in modulating inflammation in the brain

    Directory of Open Access Journals (Sweden)

    Bernd L. Fiebich

    2014-09-01

    Full Text Available Brain inflammation is a common occurrence following responses to varied insults such as bacterial infections, stroke, traumatic brain injury and neurodegenerative disorders. A common mediator for these varied inflammatory responses is prostaglandin E2 (PGE2, produced by the enzymatic activity of cyclooxygenases (COX 1 and 2. Previous attempts to reduce neuronal inflammation through COX inhibition, by use of nonsteroidal anti-inflammatory drugs (NSAIDs, have met with limited success. We are proposing the two-hit model for neuronal injury – an initial localized inflammation mediated by PGE2 (first hit and the simultaneous release of adenosine triphosphate (ATP by injured cells (second hit, which significantly enhances the inflammatory response through increased synthesis of PGE2. Several evidences on the role of exogenous ATP in inflammation have been reported, including contrary instances where extracellular ATP reduces inflammatory events. In this review, we will examine the current literature on the role of P2 receptors, to which ATP binds, in modulating inflammatory reactions during neurodegeneration. Targeting the P2 receptors, therefore, provides a therapeutic alternative to reduce inflammation in the brain. P2 receptor-based anti-inflammatory drugs (PBAIDs will retain the activities of essential COX enzymes, yet will significantly reduce neuroinflammation by decreasing the enhanced production of PGE2 by extracellular ATP.

  20. Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

    Directory of Open Access Journals (Sweden)

    Junnan Xu

    2014-01-01

    Full Text Available This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+ after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

  1. Role of Glycogenolysis in Stimulation of ATP Release from Cultured Mouse Astrocytes by Transmitters and High K+ Concentrations

    Directory of Open Access Journals (Sweden)

    Junnan Xu

    2013-12-01

    Full Text Available This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+ after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+ -dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

  2. Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations.

    Science.gov (United States)

    Xu, Junnan; Song, Dan; Bai, Qiufang; Zhou, Lijun; Cai, Liping; Hertz, Leif; Peng, Liang

    2014-01-13

    This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

  3. Species and agonist dependent zinc modulation of endogenous and recombinant ATP-gated P2X7 receptors.

    Science.gov (United States)

    Moore, Samantha F; Mackenzie, Amanda B

    2008-12-15

    Zinc (Zn2+) and copper (Cu2+) are key signalling molecules in the immune system and regulate the activity of many ion channels. Both Zn2+ and Cu2+ potently inhibit rat P2X7 receptors via a binding site identified by mutagenesis. Here we show that extracellular Cu2+ also potently inhibits mouse P2X7 receptors. By contrast, the receptor expression system and agonist strongly influence the action of extracellular Zn2+ at mouse P2X7 receptors. Consistent with previous reports, Zn2+ inhibits recombinant rat P2X7 receptors. However, recombinant mouse P2X7 receptors are potentiated by Zn2+ when activated by ATP4- but inhibited when stimulated with the ATP analogue BzATP4-. Endogenous murine macrophage P2X7 receptors are not modulated by Zn2+ when stimulated by ATP4- however Zn2+ inhibits BzATP4- mediated responses. In summary, these findings provide a fundamental insight into the differential actions of Zn2+ and Cu2+ between different P2X7 receptor species.

  4. P2X receptor-mediated ATP purinergic signaling in health and disease

    Directory of Open Access Journals (Sweden)

    Jiang LH

    2012-09-01

    Full Text Available Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP, an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

  5. Customized ATP towpreg. [Automated Tow Placement

    Science.gov (United States)

    Sandusky, Donald A.; Marchello, Joseph M.; Baucom, Robert M.; Johnston, Norman J.

    1992-01-01

    Automated tow placement (ATP) utilizes robotic technology to lay down adjacent polymer-matrix-impregnated carbon fiber tows on a tool surface. Consolidation and cure during ATP requires that void elimination and polymer matrix adhesion be accomplished in the short period of heating and pressure rolling that follows towpreg ribbon placement from the robot head to the tool. This study examined the key towpreg ribbon properties and dimensions which play a significant role in ATP. Analysis of the heat transfer process window indicates that adequate heating can be achieved at lay down rates as high as 1 m/sec. While heat transfer did not appear to be the limiting factor, resin flow and fiber movement into tow lap gaps could be. Accordingly, consideration was given to towpreg ribbon having uniform yet non-rectangular cross sections. Dimensional integrity of the towpreg ribbon combined with customized ribbon architecture offer great promise for processing advances in ATP of high performance composites.

  6. An RNA motif that binds ATP

    Science.gov (United States)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  7. Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion

    DEFF Research Database (Denmark)

    Mortensen, Stefan P.; Gonzalez-Alonso, Jose; Nielsen, Jens Jung

    2009-01-01

    ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanism remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on limb muscle interstitial...... ATP and NE concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and norepinephrine (NE) concentrations were measured during: 1) femoral arterial ATP infusion (0.......42+/-0.04 and 2.26+/-0.52 mumol/min; mean+/-SEM) and 2) one-leg knee-extensor exercise (18+/-0 and 37+/-2W) in 10 healthy, male subjects. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from ~0.3 L/min at baseline to 4.2+/-0.3 and 4.6+/-0.5 L/min, respectively, whereas...

  8. The role of individual domains and the significance of shedding of ATP6AP2/(prorenin receptor in vacuolar H(+-ATPase biogenesis.

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    Kenichiro Kinouchi

    Full Text Available The ATPase 6 accessory protein 2 (ATP6AP2/(prorenin receptor (PRR is essential for the biogenesis of active vacuolar H(+-ATPase (V-ATPase. Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD and transmembrane domain (TM of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M and causes X-linked mental retardation Hedera type (MRXSH and X-linked parkinsonism with spasticity (XPDS in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  9. The role of individual domains and the significance of shedding of ATP6AP2/(pro)renin receptor in vacuolar H(+)-ATPase biogenesis.

    Science.gov (United States)

    Kinouchi, Kenichiro; Ichihara, Atsuhiro; Sano, Motoaki; Sun-Wada, Ge-Hong; Wada, Yoh; Ochi, Hiroki; Fukuda, Toru; Bokuda, Kanako; Kurosawa, Hideaki; Yoshida, Naohiro; Takeda, Shu; Fukuda, Keiichi; Itoh, Hiroshi

    2013-01-01

    The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H(+)-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  10. Electrophysiology of autonomic neuromuscular transmission involving ATP.

    Science.gov (United States)

    Sneddon, P

    2000-07-01

    Electrophysiological investigations of autonomic neuromuscular transmission have provided great insights into the role of ATP as a neurotransmitter. Burnstock and Holman made the first recordings of excitatory junction potentials (e.j.p.s) produced by sympathetic nerves innervating the smooth muscle of the guinea-pig vas deferens. This led to the identification of ATP as the mediator of e.j.p.s in this tissue, where ATP acts as a cotransmitter with noradrenaline. The e.j.p.s are mediated solely by ATP acting on P2X(1) receptors leading to action potentials and a rapid phasic contraction, whilst noradrenaline mediates a slower, tonic contraction which is not dependent on membrane depolarisation. Subsequent electrophysiological studies of the autonomic innervation of smooth muscles of the urogenital, gastrointestinal and cardiovascular systems have revealed a similar pattern of response, where ATP mediates a fast electrical and mechanical response, whilst another transmitter such as noradrenaline, acetylcholine, nitric oxide or a peptide mediates a slower response. The modulation of junction potentials by a variety of pre-junctional receptors and the mechanism of inactivation of ATP as a neurotransmitter will also be described.

  11. Adenosine-5'-triphosphate (ATP supplementation improves low peak muscle torque and torque fatigue during repeated high intensity exercise sets

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    Rathmacher John A

    2012-10-01

    Full Text Available Abstract Background Intracellular concentrations of adenosine-5’-triphosphate (ATP are many times greater than extracellular concentrations (1–10 mM versus 10–100 nM, respectively and cellular release of ATP is tightly controlled. Transient rises in extracellular ATP and its metabolite adenosine have important signaling roles; and acting through purinergic receptors, can increase blood flow and oxygenation of tissues; and act as neurotransmitters. Increased blood flow not only increases substrate availability but may also aid in recovery through removal of metabolic waste products allowing muscles to accomplish more work with less fatigue. The objective of the present study was to determine if supplemental ATP would improve muscle torque, power, work, or fatigue during repeated bouts of high intensity resistance exercise. Methods Sixteen participants (8 male and 8 female; ages: 21–34 years were enrolled in a double-blinded, placebo-controlled study using a crossover design. The participants received either supplemental ATP (400 mg/d divided into 2 daily doses or placebo for 15 d. After an overnight fast, participants underwent strength and fatigue testing, consisting of 3 sets of 50 maximal knee extensions performed on a Biodex® leg dynamometer. Results No differences were detected in high peak torque, power, or total work with ATP supplementation; however, low peak torque in set 2 was significantly improved (p Conclusions Supplementation with 400 mg ATP/d for 15 days tended to reduce muscle fatigue and improved a participant’s ability to maintain a higher force output at the end of an exhaustive exercise bout.

  12. Modeling interactions among individual P2 receptors to explain complex response patterns over a wide range of ATP concentrations

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    Shu Xing

    2016-07-01

    Full Text Available EExtracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6. In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1/2 in that range (e.g. P2Y4 or P2X5 exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

  13. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels

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    Go Kasuya

    2016-02-01

    Full Text Available P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn2+ ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+ potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

  14. Extracellular Matrix Proteins

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    Linda Christian Carrijo-Carvalho

    2012-01-01

    Full Text Available Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are β-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair.

  15. ATP Synthesis in the Extremely Halophilic Bacteria

    Science.gov (United States)

    Hochstein, Lawrence I.; Morrison, David (Technical Monitor)

    1994-01-01

    The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other

  16. Macula densa cell signaling involves ATP release through a maxi anion channel.

    Science.gov (United States)

    Bell, Phillip Darwin; Lapointe, Jean-Yves; Sabirov, Ravshan; Hayashi, Seiji; Peti-Peterdi, Janos; Manabe, Ken-Ichi; Kovacs, Gergely; Okada, Yasunobu

    2003-04-01

    Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl](L)) and transmit signals to the mesangial cellafferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch-clamp studies of macula densa cells, we identified an [NaCl](L)-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 microM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl](L), by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca(2+) concentration to extracellular application of ATP (EC(50) 0.8 microM). Importantly, we also found increases in cytosolic Ca(2+) concentration with elevations in [NaCl](L), when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y(2) receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

  17. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

    DEFF Research Database (Denmark)

    Ostergaard, L; Teilum, K; Mirza, O

    2000-01-01

    to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover......, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure...... of ATP A2 was solved to 1.45 A resolution at 100 K. Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex. The analysis indicates that the precursors p...

  18. Synthetic peptides target ATP translocase of ‘Candidatus Liberibacter asiaticus’ to block ATP uptake

    Science.gov (United States)

    As an obligate intracellular pathogen, ‘Candidatus Liberibacter asiaticus’ (Las) may act as an “energy parasite” by importing ATP from its host’s cells. We previously demonstrated that the Las translocase NttA (gb|ACX71867.1) is functional in Escherichia coli and enables the direct import of ATP/ADP...

  19. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this st......Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim...... of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either...... release of both ATP-consuming and ATP-generating enzymes into pancreatic juice. This newly discovered richness of secreted enzymes underscores the importance of purine signaling between acini and pancreatic ducts lumen and implies regulation of the purine-converting enzymes release....

  20. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  1. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  2. Extracellular matrix structure.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented.

  3. ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation.

    Science.gov (United States)

    Riteau, N; Baron, L; Villeret, B; Guillou, N; Savigny, F; Ryffel, B; Rassendren, F; Le Bert, M; Gombault, A; Couillin, I

    2012-10-11

    Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.

  4. Extracellular adenosine 5'-triphosphate and lipopolysaccharide induce interleukin-1β release in canine blood.

    Science.gov (United States)

    Spildrejorde, Mari; Curtis, Stephen J; Curtis, Belinda L; Sluyter, Ronald

    2014-01-15

    Binding of extracellular adenosine 5'-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1β in humans and mice. However, the release of IL-1β in dogs remains poorly defined. Using a canine IL-1β enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1β release in a canine blood-based assay. Short-term incubations (30 min) with ATP induced IL-1β release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1β release from blood not primed with LPS. ATP-induced IL-1β release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24h) with LPS induced IL-1β release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1β release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1β release in dogs, and that ATP- but not LPS-induced IL-1β release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1β release in dogs.

  5. ATP induces P2X7 receptor-independent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Koch-Nolte, Friedrich; Haag, Friedrich; Bulfone-Paus, Silvia

    2009-04-01

    Extracellular ATP mediates a diverse array of biological responses in many cell types and tissues, including immune cells. We have demonstrated that ATP induces purinergic receptor P2X(7) mediated membrane permeabilization, apoptosis, and cytokine expression in murine mast cells (MCs). Here, we report that MCs deficient in the expression of the P2X(7) receptor are resistant to the ATP-induced membrane permeabilization and apoptosis. However, ATP affects the tyrosine phosphorylation pattern of P2X(7)knockout cells, leading to the activation of ERK1/2. Furthermore, ATP induces expression of several cytokines and chemokines in these cells, including IL-4, IL-6, IFN-gamma, TNF-alpha, RANTES, and MIP-2, at the mRNA level. In addition, the release of IL-6 and IL-13 to cell-conditioned medium was confirmed by ELISA. The ligand selectivity and pharmacological profile indicate the involvement of two P2X family receptors, P2X(1) and P2X(3). Thus, depending on genetic background, particular tissue microenvironment, and ATP concentration, MCs can presumably engage different P2X receptor subtypes, which may result in functionally distinct biological responses to extracellular nucleotides. This finding highlights a novel level of complexity in the sophisticated biology of MCs and may facilitate the development of new therapeutic approaches to modulate MC activities.

  6. Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes.

    Science.gov (United States)

    Kamidate, Tamio; Yanashita, Kenji; Tani, Hirofumi; Ishida, Akihiko; Notani, Mizuyo

    2006-01-01

    Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an ATP extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes.

  7. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    Science.gov (United States)

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  8. Electrophysiological effects of ATP on brain neurones.

    Science.gov (United States)

    Illes, P; Nieber, K; Nörenberg, W

    1996-12-01

    1. The electrophysiological effects of ATP on brain neurones are either due to the direct activation of P2 purinoceptors by the unmetabolized nucleotide or to the indirect activation of P1. purinoceptors by the degradation product adenosine. 2. Two subtypes of P2 purinoceptors are involved, a ligand-activated ion channel (P2X) and a G protein-coupled receptor (P2Y). Hence, the stimulation of P2X purinoceptors leads to a cationic conductance increase, while the stimulation of P2Y purinoceptors leads to a G protein-mediated opening or closure of potassium channels. 3. ATP may induce a calcium-dependent potassium current by increasing the intracellular Ca2+ concentration. This is due either to the entry of Ca2+ via P2X purinoceptors or to the activation of metabotropic P2Y purinoceptors followed by signaling via the G protein/phospholipase C/inositol 1,4,5-trisphosphate (IP3) cascade. Eventually, IP3 releases Ca2+ from its intracellular pools. 4. There is no convincing evidence for the presence of P2U purinoceptors sensitive to both ATP and UTP, or pyrimidinoceptors sensitive to UTP only, in the central nervous system (CNS). 5. ATP-sensitive P2X and P2Y purinoceptors show a wide distribution in the CNS and appear to regulate important neuronal functions.

  9. Torque generation mechanism of ATP synthase

    Science.gov (United States)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  10. Calcium and ATP control multiple vital functions

    Science.gov (United States)

    Verkhratsky, Alexei

    2016-01-01

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca2+ concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca2+ concentration (which in all life forms is kept around 50–100 nM) forms the basis for a universal intracellular signalling system in which Ca2+ acts as a second messenger. Maintenance of transmembrane Ca2+ gradients, in turn, requires ATP-dependent Ca2+ transport, thus further emphasizing the inseparable links between these two substances. Ca2+ signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca2+ signalling relies on cell specific Ca2+ signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca2+ signalling toolkits lead to aberrant Ca2+ signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377728

  11. Seahorse Xfe 24 Extracellular Flux Analyzer-Based Analysis of Cellular Respiration in Caenorhabditis elegans.

    Science.gov (United States)

    Luz, Anthony L; Smith, Latasha L; Rooney, John P; Meyer, Joel N

    2015-11-02

    Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF(e) 24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans.

  12. Autocrine regulation of TGF-β1-induced cell migration by exocytosis of ATP and activation of P2 receptors in human lung cancer cells.

    Science.gov (United States)

    Takai, Erina; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Sawada, Keisuke; Moriyama, Yoshinori; Kojima, Shuji

    2012-11-01

    TGF-β1 plays a key role in cancer progression through induction of various biological effects, including cell migration. Extracellular nucleotides, such as ATP, released from cells play a role in signaling through activation of P2 receptors. We show here that exocytosis of ATP followed by activation of P2 receptors play a key role in TGF-β1-induced actin remodeling associated with cell migration. Treatment with TGF-β1 facilitated migration of human lung cancer A549 cells, which was blocked by pretreatment with ecto-nucleotidase and P2 receptor antagonists. ATP and P2 agonists facilitated cell migration. TGF-β1-induced actin remodeling, which contributes to cell migration, was also suppressed by pretreatment with ecto-nucleotidase and P2 receptor antagonists. Knockdown of P2X7 receptor suppressed TGF-β1-induced migration and actin remodeling. These results indicate the involvement of TGF-β1-induced ATP release in cell migration, at least in part, through activation of P2X7 receptors. TGF-β1 caused release of ATP from A549 cells within 10 minutes. Both ATP-enriched vesicles and expression of a vesicular nucleotide transporter (VNUT) SLC17A9, which is responsible for exocytosis of ATP, were found in cytosol of A549 cells. TGF-β1 failed to induce release of ATP from SLC17A9-knockdown cells. TGF-β1-induced cell migration and actin remodeling were also decreased in SLC17A9-knockdown cells. These results suggest the importance of exocytosis of ATP in cell migration. We conclude that autocrine signaling through exocytosis of ATP and activation of P2 receptors is required for the amplification of TGF-β1-induced migration of lung cancer cells.

  13. ATP measurements for monitoring microbial drinking water quality

    DEFF Research Database (Denmark)

    Vang, Óluva Karin

    methods are vital for an improved surveillance and distribution of clean and safe drinking water. One of these rapid methods is the ATP assay. This thesis encompasses various methodological aspects of the ATP assay describing the principal and theory of the ATP assay measurement. ATP is the main energy...... carrying molecule in living cells, thus ATP can be used as a parameter for microbial activity. ATP is extracted from cells through cell lysis and subsequently assayed with the luciferase enzyme and its substrate luciferin, resulting in bioluminescence, i.e. light emission which can be quantified....... The overall aim of this PhD study was to investigate various methodological features of the ATP assay for a potential implementation on a sensor platform as a real-time parameter for continuous on-line monitoring of microbial drinking water quality. Commercial reagents are commonly used to determine ATP...

  14. Extracellular vesicles for drug delivery

    NARCIS (Netherlands)

    Vader, Pieter; Mol, Emma A; Pasterkamp, Gerard; Schiffelers, Raymond M

    Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained

  15. Calcium signals in the nucleus accumbens: Activation of astrocytes by ATP and succinate

    Directory of Open Access Journals (Sweden)

    Emri Zsuzsa

    2011-10-01

    Full Text Available Abstract Background Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc, and thereby to explore the action of citric acid cycle intermediate succinate (SUC. Results We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43 and the glial fibrillary acidic protein (GFAP indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthioadenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 μM in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. Conclusions We

  16. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highl

  17. An ATP synthase harboring an atypical γ–subunit is involved in ATP synthesis in tomato fruit chromoplasts

    National Research Council Canada - National Science Library

    Pateraki, Irini; Renato, Marta; Azcón‐Bieto, Joaquín; Boronat, Albert

    2013-01-01

    ... and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP , and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources...

  18. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Masaki Nakano

    2017-08-01

    Full Text Available Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances, which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.

  19. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

    Science.gov (United States)

    Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

    2014-11-01

    The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

  20. Yeast ADP/ATP Carrier Isoform 2

    Science.gov (United States)

    Clémençon, Benjamin; Rey, Martial; Trézéguet, Véronique; Forest, Eric; Pelosi, Ludovic

    2011-01-01

    The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure. PMID:21868387

  1. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    Science.gov (United States)

    Brand, M D; Lehninger, A L

    1977-05-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.

  2. Activation of volume-regulated Cl− channels by ACh and ATP in Xenopus follicles

    Science.gov (United States)

    Pérez-Samartín, Alberto L; Miledi, Ricardo; Arellano, Rogelio O

    2000-01-01

    Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique.In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application.In follicles, the permeability ratios for different anions with respect to Cl− were similar for both ICl,swell and Sin, with a sequence of: SCN− > I− > Br−≥ NO3−≥ Cl− > gluconate ≥ cyclamate > acetate > SO42−.Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was ∼100% slower at pH 8.0 compared with that at pH 6.0.Lanthanides inhibited ICl,swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 ± 1.9 μm, while Sin was blocked up to 55% with an apparent IC50 of 36 ± 2.6 μm.Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 ± 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to −20 mV. This single-channel activity was increased by application of ACh or ATP.The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling.All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity

  3. Human endomembrane H sup + pump strongly resembles the ATP-synthetase of Archaebacteria

    Energy Technology Data Exchange (ETDEWEB)

    Suedhof, T.C.; Stone, D.K.; Johnston, P.A.; Xie, Xiaosong (Univ. of Texas Southwestern Medical Center, Dallas (USA)); Fried, V.A. (Saint Jude Children' s Research Hospital, Memphis, TN (USA))

    1989-08-01

    Preparations of mammalian H{sup +} pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H{sup +} pump are unknown from animal cells. The authors report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H{sup +} pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H{sup +} gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H{sup +} pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.

  4. Shockwaves increase T-cell proliferation and IL-2 expression through ATP release, P2X7 receptors, and FAK activation.

    Science.gov (United States)

    Yu, Tiecheng; Junger, Wolfgang G; Yuan, Changji; Jin, An; Zhao, Yi; Zheng, Xueqing; Zeng, Yanjun; Liu, Jianguo

    2010-03-01

    Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/mm(2) induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation.

  5. Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival.

    Science.gov (United States)

    Hucthagowder, Vishwanathan; Morava, Eva; Kornak, Uwe; Lefeber, Dirk J; Fischer, Björn; Dimopoulou, Aikaterini; Aldinger, Annika; Choi, Jiwon; Davis, Elaine C; Abuelo, Dianne N; Adamowicz, Maciej; Al-Aama, Jumana; Basel-Vanagaite, Lina; Fernandez, Bridget; Greally, Marie T; Gillessen-Kaesbach, Gabriele; Kayserili, Hulya; Lemyre, Emmanuelle; Tekin, Mustafa; Türkmen, Seval; Tuysuz, Beyhan; Yüksel-Konuk, Berrin; Mundlos, Stefan; Van Maldergem, Lionel; Wevers, Ron A; Urban, Zsolt

    2009-06-15

    Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse-chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells.

  6. Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors.

    Directory of Open Access Journals (Sweden)

    Ayumi Kawano

    Full Text Available Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2, and ATP release and interleukin (IL-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors.

  7. The distribution of ATP within tomato (Lycopersicon esculentum Mill.) embryos correlates with germination whee as total ATP concentration does not

    NARCIS (Netherlands)

    Spoelstra, P.; Joosen, R.V.L.; Hilhorst, H.W.M.

    2002-01-01

    The distribution of ATP in tomato seeds was visualized by monitoring the luminescence of frozen sections on top of a gel containing all the components of the luciferase reaction, but excluding ATP. ATP was imaged in germinating tomato seeds at intervals of 3, 6, 17, 24 and 48 h and in seeds with pri

  8. The distribution of ATP within tomato (Lycopersicon esculentum Mill.) embryos correlates with germination whee as total ATP concentration does not

    NARCIS (Netherlands)

    Spoelstra, P.; Joosen, R.V.L.; Hilhorst, H.W.M.

    2002-01-01

    The distribution of ATP in tomato seeds was visualized by monitoring the luminescence of frozen sections on top of a gel containing all the components of the luciferase reaction, but excluding ATP. ATP was imaged in germinating tomato seeds at intervals of 3, 6, 17, 24 and 48 h and in seeds with

  9. Mapping the interactions between ATP and the sarcoplasmic reticulum Ca 2 + -ATPase with ATP and ATP analogs studied by Fourier transform infrared spectroscopy

    OpenAIRE

    Liu, Man

    2004-01-01

    Die Infrarotspektroskopie in Verbindung mit photoaktivierbaren Substraten wurde zur Untersuchung von Substrat-Protein-Wechselwirkungen eingesetzt. Dabei wurden Konformationsänderungen der Ca2+-ATPase des Sarkoplasmatischen Retikulums bei Bindung des Nukleotids, der Phosphorylierung der ATPase und der Hydrolyse des Phosphoenzyms beobachtet. Verwender wurden das native Substrat ATP und seine Analoga ADP, AMPPNP, 2'-deoxyATP, 3'-deoxyATP, ITP, AMP, Pyrophosphat, Ribosetriphosphat und TNP-AMP beo...

  10. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    Science.gov (United States)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  11. Ratiometric detection of adenosine triphosphate (ATP) in water and real-time monitoring of apyrase activity with a tripodal zinc complex.

    Science.gov (United States)

    Butler, Stephen J

    2014-11-24

    Two tripodal fluorescent probes Zn⋅L(1,2) have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn⋅L(1) allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of several competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid.

  12. Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop.

    Science.gov (United States)

    Michalski, Kevin; Kawate, Toshimitsu

    2016-02-01

    Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating.

  13. Clostridium pasteurianum F1Fo ATP Synthase: Operon, Composition, and Some Properties

    OpenAIRE

    2003-01-01

    The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced. It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ɛ), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of ATPase activity in the membranes of C. pasteurianum was low compared to what ha...

  14. Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites.

    Science.gov (United States)

    Coutinho-Silva, Robson; Ojcius, David M

    2012-11-01

    Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1β (IL-1β), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.

  15. Metabolic requirements for neutrophil extracellular traps formation

    Science.gov (United States)

    Rodríguez-Espinosa, Oscar; Rojas-Espinosa, Oscar; Moreno-Altamirano, María Maximina Bertha; López-Villegas, Edgar Oliver; Sánchez-García, Francisco Javier

    2015-01-01

    As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 1010 to 1011 cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis. PMID:25545227

  16. Identification of the inorganic pyrophosphate metabolizing, ATP substituting pathway in mammalian spermatozoa.

    Directory of Open Access Journals (Sweden)

    Young-Joo Yi

    Full Text Available Inorganic pyrophosphate (PPi is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1 in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.

  17. Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1β Release.

    Science.gov (United States)

    Hecker, Andreas; Küllmar, Mira; Wilker, Sigrid; Richter, Katrin; Zakrzewicz, Anna; Atanasova, Srebrena; Mathes, Verena; Timm, Thomas; Lerner, Sabrina; Klein, Jochen; Kaufmann, Andreas; Bauer, Stefan; Padberg, Winfried; Kummer, Wolfgang; Janciauskiene, Sabina; Fronius, Martin; Schweda, Elke K H; Lochnit, Günter; Grau, Veronika

    2015-09-01

    IL-1β is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1β plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1β release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1β synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1β by caspase-1, and release of mature IL-1β. Mechanisms controlling IL-1β release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1β release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1β and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.

  18. Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.

    Science.gov (United States)

    Habermacher, Chloé; Martz, Adeline; Calimet, Nicolas; Lemoine, Damien; Peverini, Laurie; Specht, Alexandre; Cecchini, Marco; Grutter, Thomas

    2016-01-25

    P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as 'molecular tweezers' to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

  19. ATP Cofactor energético

    OpenAIRE

    Granados Moreno, Jairo Enrique

    2014-01-01

    El documento explica la importancia de la adenosín trifosfato ó trifosfato de adenosina (ATP). Presenta los flujos y tipos de energía en animales. Estos conceptos son muy útiles a la hora de entender el flujo energético en la naturaleza pues permite comprender que en el paso de los compuestos por todos los procesos metabólicos, por ejemplo de la glucosa al CO2 hay unas 21 reacciones, se va liberando energía en forma de calor e incrementando la entropía, en definitiva

  20. Mitochondrial flashes regulate ATP homeostasis in the heart.

    Science.gov (United States)

    Wang, Xianhua; Zhang, Xing; Wu, Di; Huang, Zhanglong; Hou, Tingting; Jian, Chongshu; Yu, Peng; Lu, Fujian; Zhang, Rufeng; Sun, Tao; Li, Jinghang; Qi, Wenfeng; Wang, Yanru; Gao, Feng; Cheng, Heping

    2017-07-10

    The maintenance of a constant ATP level ('set-point') is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart.

  1. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

    Science.gov (United States)

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

    2013-01-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  2. Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

    Science.gov (United States)

    Blodgett, David M.; De Zutter, Julie K.; Levine, Kara B.; Karim, Pusha; Carruthers, Anthony

    2007-01-01

    Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7. PMID:17635959

  3. Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin.

    Science.gov (United States)

    Valentin-Ranc, C; Carlier, M F

    1991-04-25

    The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.

  4. Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif.

    Science.gov (United States)

    Tu, Q; Yu, L; Zhang, P; Zhang, M; Zhang, H; Jiang, J; Chen, C; Zhao, S

    2000-04-01

    A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome.

  5. Extracellular vesicles for drug delivery

    NARCIS (Netherlands)

    Vader, Pieter; Mol, Emma A; Pasterkamp, Gerard; Schiffelers, Raymond M

    2016-01-01

    Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained consider

  6. Porphyromonas gingivalis fimbriae dampen P2X7-dependent IL-1β secretion

    Science.gov (United States)

    Morandini, Ana Carolina; Ramos-Junior, Erivan S.; Potempa, Jan; Nguyen, Ky-Anh; Oliveira, Ana Carolina; Bellio, Maria; Ojcius, David M.; Scharfstein, Julio; Coutinho-Silva, Robson

    2014-01-01

    Porphyromonas gingivalis is a major contributor to the pathogenesis of periodontitis, an infection-driven inflammatory disease that leads to bone destruction. This pathogen stimulates pro-IL-1β synthesis but not mature IL-1β secretion, unless the P2X7 receptor is activated by extracellular ATP. Here, we investigated the role of Pg fimbriae in eATP-induced IL-1β release. Bone marrow derived macrophages (BMDMs) from wild type (WT) or P2X7-deficient mice were infected with Pg (strain 381) or isogenic fimbriae deficient (strain DPG3) with or without subsequent eATP stimulation. DPG3 induced higher IL-1β secretion after eATP stimulation compared to 381 in WT BMDMs, but not in P2X7-deficient cells. This mechanism was dependent of K+ efflux and Ca2+-iPLA2 activity. Accordingly, non-fimbriated Pg failed to inhibit apoptosis via eATP/P2X7-pathway. Furthermore, Pg-driven stimulation of IL-1β was TLR2- and MyD88-dependent, and irrespective of fimbriae expression. Fimbriae-dependent down-modulation of IL-1β was selective, as levels of other cytokines remained unaffected by P2X7 deficiency. Confocal microscopy demonstrated the presence of discrete P2X7 expression in the absence of Pg stimulation which was enhanced by 381-stimulated cells. Notably, DPG3-infected macrophages revealed a distinct pattern of P2X7 receptor expression with a markedly foci formation. Collectively, these data demonstrate that eATP-induced IL-1β secretion is impaired by Pg fimbriae in a P2X7-dependent manner. PMID:24925032

  7. ATP inhibits the generation and function of regulatory T cells through the activation of purinergic P2X receptors.

    Science.gov (United States)

    Schenk, Ursula; Frascoli, Michela; Proietti, Michele; Geffers, Robert; Traggiai, Elisabetta; Buer, Jan; Ricordi, Camillo; Westendorf, Astrid M; Grassi, Fabio

    2011-03-01

    Extracellular nucleotides are pleiotropic regulators of mammalian cell function. Adenosine triphosphate (ATP) released from CD4(+) helper T cells upon stimulation of the T cell receptor (TCR) contributes in an autocrine manner to the activation of mitogen-activated protein kinase (MAPK) signaling through purinergic P2X receptors. Increased expression of p2rx7, which encodes the purinergic receptor P2X7, is part of the transcriptional signature of immunosuppressive CD4(+)CD25(+) regulatory T cells (T(regs)). Here, we show that the activation of P2X7 by ATP inhibits the suppressive potential and stability of T(regs). The inflammatory cytokine interleukin-6 (IL-6) increased ATP synthesis and P2X7-mediated signaling in T(regs), which induced their conversion to IL-17-secreting T helper 17 (T(H)17) effector cells in vivo. Moreover, pharmacological antagonism of P2X receptors promoted the cell-autonomous conversion of naïve CD4(+) T cells into T(regs) after TCR stimulation. Thus, ATP acts as an autocrine factor that integrates stimuli from the microenvironment and cellular energetics to tune the developmental and immunosuppressive program of the T cell in adaptive immune responses.

  8. Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas.

    Science.gov (United States)

    Drapier, D.; Girard-Bascou, J.; Wollman, F. A.

    1992-03-01

    We analyzed three nuclear mutants of Chlamydomonas reinhardtii altered in the expression of the chloroplast genes atpA or atpB coding for the [alpha] or [beta] subunit of the chloroplast ATP synthase. These mutants revealed the existence of three nuclear products controlling the expression of the two chloroplast genes: the first one acts on the translation of the atpA transcript, and the two others act specifically on the stability of either the atpB or the atpA mRNAs. The nuclear mutation responsible for the decreased stability of the atpB mRNA prevented translation of the corresponding polypeptide. In contrast, the mutation responsible for the decreased stability of the atpA mRNA had limited effect on the translation of the [alpha] subunit, thereby allowing its accumulation and assembly in an active ATP synthase. Although acting originally on the expression of only one of the two main coupling factor 1 subunits, the three mutations caused a change in the translation rate of the other subunit, as viewed in 5-min pulse labeling experiments. This is indicative of a concerted expression of the [alpha] and [beta] subunits at an early post-translational step, or during translation, that may be critical for the assembly of the chloroplast ATP synthase.

  9. The action of selenite on ATP synthesis in rat lens

    OpenAIRE

    Adamchak, Marsha Ann

    1986-01-01

    A subcutaneous injection of sodium selenite (30 umol/kg body weight) in 10â day old rats produced a cataract within 72 hours. Lens opacification was preceded by a 15% decrease in ATP content. Lens ATP did not fully recover to control concentrations by 11 days postâ injection. A moderate correlation existed between lens weight and total ATP content in control lenses

  10. Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity

    Directory of Open Access Journals (Sweden)

    Francesca Schena

    2013-06-01

    Full Text Available Immunoglobulin (Ig isotype diversification by class switch recombination (CSR is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID. Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca2+-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5′-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

  11. Intracellular Assessment of ATP Levels in Caenorhabditis elegans

    Science.gov (United States)

    Palikaras, Konstantinos; Tavernarakis, Nektarios

    2017-01-01

    Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans. PMID:28194429

  12. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  13. Dose-dependent ATP depletion and cancer cell death following calcium electroporation, relative effect of calcium concentration and electric field strength

    DEFF Research Database (Denmark)

    Hansen, Emilie Louise; Sozer, Esin Bengisu; Romeo, Stefania;

    2015-01-01

    death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy. METHODS: In three human cell lines--H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability...... was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells. RESULTS: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p...-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field. GENERAL SIGNIFICANCE: This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials....

  14. Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

    Science.gov (United States)

    Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

  15. Respiratory infections cause the release of extracellular vesicles: implications in exacerbation of asthma/COPD.

    Directory of Open Access Journals (Sweden)

    Suffwan Eltom

    Full Text Available Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1β/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1β/IL-18, neutrophilia and subsequent disease exacerbations.To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis.Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1β/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia.This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.

  16. Modelling the ATP production in mitochondria

    CERN Document Server

    Saa, Alberto

    2012-01-01

    We revisit here the mathematical model for ATP production in mitochondria introduced recently by Bertram, Pedersen, Luciani, and Sherman (BPLS) as a simplification of the more complete but intricate Magnus and Keizer's model. We correct some inaccuracies in the BPLS original approximations and then analyze some of the dynamical properties of the model. We infer from exhaustive numerical explorations that the enhanced BPLS equations have a unique attractor fixed point for physiologically acceptable ranges of mitochondrial variables and respiration inputs. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium ${\\rm Ca}_{\\rm c}$ and the substrate fructose 1,6-bisphosphate FBP. The same effect of calcium saturation reported for the original BPLS model is observed here. We find out, however, an interesting non-stationary effect: the inertia of the model tends to increase considerably for high concentrations of ...

  17. Extracellular vesicles: Exosomes, microvesicles, and friends

    NARCIS (Netherlands)

    Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

    2013-01-01

    Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

  18. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  19. ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase

    Science.gov (United States)

    Hochstein, L. I.

    1992-01-01

    Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

  20. Neutrophil Extracellular Traps and Microcrystals

    Science.gov (United States)

    2017-01-01

    Neutrophil extracellular traps represent a fascinating mechanism by which PMNs entrap extracellular microbes. The primary purpose of this innate immune mechanism is thought to localize the infection at an early stage. Interestingly, the ability of different microcrystals to induce NET formation has been recently described. Microcrystals are insoluble crystals with a size of 1–100 micrometers that have different composition and shape. Microcrystals have it in common that they irritate phagocytes including PMNs and typically trigger an inflammatory response. This review is the first to summarize observations with regard to PMN activation and NET release induced by microcrystals. Gout-causing monosodium urate crystals, pseudogout-causing calcium pyrophosphate dehydrate crystals, cholesterol crystals associated with atherosclerosis, silicosis-causing silica crystals, and adjuvant alum crystals are discussed. PMID:28373994

  1. Neutrophil Extracellular Traps and Microcrystals.

    Science.gov (United States)

    Rada, Balázs

    2017-01-01

    Neutrophil extracellular traps represent a fascinating mechanism by which PMNs entrap extracellular microbes. The primary purpose of this innate immune mechanism is thought to localize the infection at an early stage. Interestingly, the ability of different microcrystals to induce NET formation has been recently described. Microcrystals are insoluble crystals with a size of 1-100 micrometers that have different composition and shape. Microcrystals have it in common that they irritate phagocytes including PMNs and typically trigger an inflammatory response. This review is the first to summarize observations with regard to PMN activation and NET release induced by microcrystals. Gout-causing monosodium urate crystals, pseudogout-causing calcium pyrophosphate dehydrate crystals, cholesterol crystals associated with atherosclerosis, silicosis-causing silica crystals, and adjuvant alum crystals are discussed.

  2. Neutrophil Extracellular Traps and Microcrystals

    Directory of Open Access Journals (Sweden)

    Balázs Rada

    2017-01-01

    Full Text Available Neutrophil extracellular traps represent a fascinating mechanism by which PMNs entrap extracellular microbes. The primary purpose of this innate immune mechanism is thought to localize the infection at an early stage. Interestingly, the ability of different microcrystals to induce NET formation has been recently described. Microcrystals are insoluble crystals with a size of 1–100 micrometers that have different composition and shape. Microcrystals have it in common that they irritate phagocytes including PMNs and typically trigger an inflammatory response. This review is the first to summarize observations with regard to PMN activation and NET release induced by microcrystals. Gout-causing monosodium urate crystals, pseudogout-causing calcium pyrophosphate dehydrate crystals, cholesterol crystals associated with atherosclerosis, silicosis-causing silica crystals, and adjuvant alum crystals are discussed.

  3. Extracellular secretion of recombinant proteins

    Science.gov (United States)

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  4. The evidence for association of ATP2B2 polymorphisms with autism in Chinese Han population.

    Directory of Open Access Journals (Sweden)

    Wen Yang

    Full Text Available BACKGROUND: Autism is a neurodevelopmental disorder with a high estimated heritability. ATP2B2, located on human chromosome 3p25.3, encodes the plasma membrane calcium-transporting ATPase 2 which extrudes Ca(2+ from cytosol into extracellular space. Recent studies reported association between ATP2B2 and autism in samples from Autism Genetic Resource Exchange (AGRE and Italy. In this study, we investigated whether ATP2B2 polymorphisms were associated with autism in Chinese Han population. METHODS: We performed a family based association study between five SNPs (rs35678 in exon, rs241509, rs3774180, rs3774179, and rs2278556 in introns in ATP2B2 and autism in 427 autism trios of Han Chinese descent. All SNPs were genotyped using the Sequenom genotyping platform. The family-based association test (FBAT program was used to perform association test for SNPs and haplotype analyses. RESULTS: This study demonstrated a preferential transmission of T allele of rs3774179 to affected offsprings under an additive model (T>C, Z = 2.482, p = 0.013. While C allele of rs3774179 showed an undertransmission from parents to affected children under an additive and a dominant model, respectively (Z = -2.482, p = 0.013; Z = -2.591, p = 0.0096. Haplotype analyses revealed that three haplotypes were significantly associated with autism. The haplotype C-C (rs3774180-rs3774179 showed a significant undertransmission from parents to affected offsprings both in specific and global haplotype FBAT (Z = -2.037, p = 0.042; Global p = 0.03. As for the haplotype constructed by rs3774179 and rs2278556, C-A might be a protective haplotype (Z = -2.206, p = 0.027; Global p = 0.04, while T-A demonstrated an excess transmission from parents to affected offsprings (Z = 2.143, p = 0.032. These results were still significant after using the permutation method to obtain empirical p values. CONCLUSIONS: Our research suggested that ATP2B2

  5. Subunit movements in single membrane-bound H+-ATP synthases from chloroplasts during ATP synthesis.

    Science.gov (United States)

    Bienert, Roland; Rombach-Riegraf, Verena; Diez, Manuel; Gräber, Peter

    2009-12-25

    Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

  6. ATP storage and uptake by isolated pancreatic zymogen granules

    DEFF Research Database (Denmark)

    Haanes, Kristian Agmund; Novak, Ivana

    2010-01-01

    ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism...... for ATP transport into the ZG. ZG were isolated and the ATP content was measured using luciferin/luciferase assays and was related to protein in the sample. The estimate of ATP concentration in freshly isolated granules was 40-120 µM. The ATP uptake had an apparent Km value of 4.9±2.1 mM when granules...... were incubated without Mg2+ and a Km value of 0.47±0.05 mM in the presence of Mg2+, both in pH 6.0 buffers. The uptake of ATP was significantly higher at pH 7.2 compared with pH 6.0 solutions. The anion transport blockers DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate) and Evans Blue inhibited ATP...

  7. K ATP channels in pig and human intracranial arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Sørensen, Mette Aaskov; Strøbech, Lotte Bjørg

    2008-01-01

    Clinical trials suggest that synthetic ATP-sensitive K(+) (K(ATP)) channel openers may cause headache and migraine by dilating cerebral and meningeal arteries. We studied the mRNA expression profile of K(ATP) channel subunits in the pig and human middle meningeal artery (MMA) and in the pig middle...... cerebral artery (MCA). We determined the order of potency of four K(ATP) channel openers when applied to isolated pig MMA and MCA, and we examined the potential inhibitory effects of the Kir6.1 subunit specific K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the isolated...... pig MMA and MCA. Using conventional RT-PCR, we detected the mRNA transcripts of the K(ATP) channel subunits Kir6.1 and SUR2B in all the examined pig and human intracranial arteries. Application of K(ATP) channel openers to isolated pig MMA and MCA in myographs caused a concentration...

  8. Exon duplications in the ATP7A gene

    DEFF Research Database (Denmark)

    Mogensen, Mie; Skjørringe, Tina; Kodama, Hiroko

    2011-01-01

    BACKGROUND: Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene...

  9. Electrochemical sensing of ATP with synthetic cyclophane as recognition element

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A new electrochemical sensor for ATP with synthetic cyclophane stably attached onto single-walled carbon nanotubes (SWNTs) as the recognition elements is described. UV-vis and cyclic voltammetric results demonstrate that ATP may interact with the synthetic cyclophane recognition elements to form a stable adduct mainly through electrostatic, π-π stacking and donor-acceptor interactions. Such interactions eventually lead to a decrease in the peak currents of the cyclophane recognition elements attached onto the SWNT electronic transducer, which could be used for electrochemical sensing of ATP. Under the conditions employed here, the ratio of the decrease in the anodic peak current is linear with ATP concentration within a concentration range from 10 to 120 μM with a linear coefficiency of 0.993. This study may offer a new and simple electrochemical approach for effective sensing of ATP.

  10. ATP-sulfurylase, sulfur-compounds and plant stress tolerance

    Directory of Open Access Journals (Sweden)

    Naser A. Anjum

    2015-04-01

    Full Text Available Sulfur (S stands fourth in the list of major plant nutrients after N, P and K. Sulfate (SO42-, a form of soil-S taken up by plant roots is metabolically inert. As the first committed step of S-assimilation, ATP-sulfurylase (ATP-S catalyzes SO42--activation and yields activated high-energy compound adenosine-5′-phosphosulfate (APS that is reduced to sulfide (S2- and incorporated into cysteine (Cys. In turn, Cys acts as a precursor or donor of reduced S for a range of S-compounds such as methionine (Met, glutathione (GSH, homo-GSH (h-GSH and phytochelatins (PCs. Among S-compounds, GSH, h-GSH and PCs are known for their involvement in plant tolerance to varied abiotic stresses, Cys is a major component of GSH, h-GSH and PCs; whereas, several key stress-metabolites such as ethylene, are controlled by Met through its first metabolite S-adenosylmethionine. With the major aim of briefly highlighting S-compound-mediated role of ATP-S in plant stress tolerance, this paper: (a overviews ATP-S structure/chemistry and occurrence, (b appraises recent literature available on ATP-S roles and regulations, and underlying mechanisms in plant abiotic and biotic stress tolerance, (c summarizes ATP-S-intrinsic regulation by major S-compounds, and (d highlights major open-questions in the present context. Future research in the current direction can be devised based on the outcomes of the discussion.

  11. Nanomolar ambient ATP decelerates P2X3 receptor kinetics.

    Science.gov (United States)

    Grote, Alexander; Hans, Michael; Boldogkoi, Zsolt; Zimmer, Andreas; Steinhäuser, Christian; Jabs, Ronald

    2008-12-01

    Homomeric P2X receptors differ in their electrophysiological and pharmacological profiles. The rapidly activating and desensitizing P2X3 receptors are known for their involvement in pain signalling pathways. Modulatory effects on P2X3 receptors have been reported for low concentrations of ATP ([ATP]). This includes both, enhancement and reduction of receptor currents. The first has been reported to be mediated by activation of ectoprotein kinases and high affinity desensitization (HAD), respectively. Both processes influence receptor current amplitudes. Here we describe a new phenomenon, the modulatory influence of ambient low [ATP] on P2X3 receptor kinetics. First, we studied in HEK cells whether persistent ATP affects current decay. To this end, P2X3 receptor mediated currents, elicited by pressure application of saturating [ATP], were analyzed after pre-application of low [ATP]. Second, UV-flash photolysis of ATP was employed to investigate whether submicromolar [ATP] affects receptor activation. Finally we confirmed the action of nanomolar [ATP] on native P2X3 receptors of neurons freshly isolated from rat dorsal root ganglia. We found that persistent low [ATP] caused pronounced deceleration of receptor current activation and decay. This priming effect indicates a mechanism different from HAD. It could be explained by a pre-opening receptor isomerization, induced by the occupation of a high affinity binding site already at the resting state. The observed modulation of the receptor kinetics could be considered as a physiological fine tuning mechanism of the nociceptive system, driven by the actual ambient agonist concentration.

  12. [Evaluation of renal damage using urinary ATP analysis].

    Science.gov (United States)

    Uehara, Yuki; Yanai, Mitsuru; Kumasaka, Kazunari

    2004-10-01

    It is reported that urinary ATP concentration analysis is useful for determining urinary tract infection and renal damage caused by drugs. By means of the firefly luciferin-luciferase method, we determined the reference value of urinary free ATP and evaluated the effects of urine sediments and conditions of storage. The reference value was established as 1.77 x 10(-10) to approximately 7.70 x 10(-9)M using urine samples obtained from 63 outpatients who seemed to have no renal disease. There was no significant difference in ATP concentration between 33 males and 30 females. No significant changes were observed in 11 healthy volunteers during a 1-year period. Within-run reproducibility of ATP was satisfying (8.28% and 11.4% of coefficient value in low and high concentration samples, respectively). ATP concentration was significantly decreased after centrifugation (p < 0.05) and after filtration (p < 0.01). The amounts of the red blood cells (RBC) and white blood cells (WBC) in samples whose ATP concentration was decreased after centrifugation or filtration were significantly higher than those in samples whose concentration did not decrease (p < 0.05). Urine containing many RBCs and/or WBCs might show an artificially higher ATP concentration if no preparations has been performed. There were significant positive correlations between the ATP concentrations before and after refrigeration, but no correlations before and after freezing. It is concluded that the reference value of urinary free ATP concentration was 1.77 x 10(-10) to approximately 7.70 x 10(-9) M and that care is required in the estimation of urinary ATP concentrations in samples containing many sediments, especially with WBC and RBC.

  13. Expression of ATP7B in normal human liver

    Directory of Open Access Journals (Sweden)

    D Fanni

    2009-06-01

    Full Text Available ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.

  14. Effects of acute gamma-irradiation on extracellular adenine nucleotide hydrolysis in developing rat brain

    Science.gov (United States)

    Stanojević, I.; Drakulić, D.; Veličković, N.; Milošević, M.; Petrović, S.; Horvat, A.

    2009-09-01

    Cell membrane is highly sensitive to irradiation which, acting directly or indirectly, may disturb functions of constitutive proteins including membrane enzymes. Plasma membrane surface-located enzyme chain of ecto-nucleotide triphospho diphosphohydrolases (NTPDases) and 5'-nucleotidase are involved in termination of cell purinergic signalization by hydrolyzing extracellular, excitatory adenosine triphosphate (ATP), as well as nucleotide di-, and mono-phosphate (ADP and AMP) to neuroprotective adenosine. Extracellular ATP, ADP, and AMP hydrolyzes were examined in purified synaptic plasma membranes after whole-body acute irradiation. All measurements were done 24 h after irradiation of developing (15-, 30-day-old) and adult (90-day-old) rats with low (50 cGy) and high (2 Gy) dose of gamma-rays. Both, high and low doses inhibited nucleotide hydrolyses in 15-day-old rats; in 30-day-old rats low dose of radiation inhibited ADP and AMP hydrolyses while high dose inhibited only ATP hydrolyse. In adult rats high dose induced no effects, while low dose stimulated nucleotides hydrolyses. According to obtained results it was concluded that ecto-nucleotidases of young rats are more sensitive to irradiation, since even low dose induces inhibition of ecto-nucleotidases activities. Ionizing radiation, by decreasing brain nucleotide hydrolyses in developing rats, induces accumulation of ATP and decreases production of adenosine in synaptic cleft which could be neurocytotoxic. On the contrary, in adult rats low dose of radiation stimulates NTPDase and 5'-nucleotidase activity and protective adenosine production which indicates protective and adaptive mechanisms developed in adult brain neuronal cells.

  15. Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

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    Ip Virginia

    2010-09-01

    Full Text Available Abstract Background ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg or drug vehicle twice weekly for 8 weeks. Results In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H. High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. Conclusions In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non

  16. Adenosine Triphosphate (ATP) Inhibits Voltage-Sensitive Potassium Currents in Isolated Hensen's Cells and Nifedipine Protects Against Noise-Induced Hearing Loss in Guinea Pigs.

    Science.gov (United States)

    Ye, Rui; Liu, Jun; Jia, Zhiying; Wang, Hongyang; Wang, YongAn; Sun, Wei; Wu, Xuan; Zhao, Zhifei; Niu, Baolong; Li, Xingqi; Dai, Guanghai; Li, Jianxiong

    2016-06-13

    BACKGROUND There is increasing evidence that adenosine triphosphate (ATP), a well-known neurotransmitter and neuromodulator in the central nervous system, plays an important role as an extracellular chemical messenger in the cochlea. MATERIAL AND METHODS Using a whole-cell recording technique, we studied the effects of ATP on isolated Hensen's cells, which are supporting cells in the cochlea, to determine if they are involved in the transduction of ions with hair cells. RESULTS ATP (0.1-10 µM) reduced the potassium current (IK+) in the majority of the recorded Hensen's cells (21 out of 25 cells). An inward current was also induced by high concentrations of ATP (100 µM to 10 mM), which was reversibly blocked by 100 µM suramin (a purinergic antagonist) and blocked by nifedipine (an L-type calcium channel blocker). After the cochleas were perfused with artificial perilymph solutions containing nifedipine and exposed to noise, the amplitude increase in the compound action potential (CAP) threshold and the reduction in cochlear microphonics was lower than when they were exposed to noise alone. CONCLUSIONS Our results suggest that ATP can block IK+ channels at a low concentration and induce an inward Ca2+ current at high concentrations, which is reversed by purinergic receptors. Nifedipine may have a partially protective effect on noise-induced hearing loss (NIHL).

  17. An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts

    DEFF Research Database (Denmark)

    Pateraki, Irini; Renato, Marta; Azcõn-Bieto, Joaquín

    2013-01-01

    synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes...... in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report...... the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize...

  18. Local release of ATP into the arterial inflow and venous drainage of human skeletal muscle: insight from ATP determination with the intravascular microdialysis technique

    DEFF Research Database (Denmark)

    Mortensen, Stefan; Thaning, Pia; Nyberg, Michael Permin

    2011-01-01

    Intraluminal ATP could play an important role in the local regulation of skeletal muscle blood flow, but the stimuli that cause ATP release and the levels of plasma ATP in vessels supplying and draining human skeletal muscle remain unclear. To gain insight into the mechanisms by which ATP...... is released into plasma, we measured plasma [ATP] with the intravascular microdialysis technique at rest and during dynamic exercise (normoxia and hypoxia), passive exercise, thigh compressions and arterial ATP, tyramine and ACh infusion in a total of 16 healthy young men. Femoral arterial and venous[ATP...

  19. Mechanism of ATP release from cultured marginal cells of stria vascularis in neonatal rat%新生大鼠耳蜗血管纹缘细胞释放ATP的机制

    Institute of Scientific and Technical Information of China (English)

    彭娅婷; 杨军

    2012-01-01

    目的 证实体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,并进一步探讨缘细胞释放ATP的机制.方法 分离、培养新生大鼠耳蜗血管纹缘细胞,采用生物发光法分别检测巴佛洛霉素A1、己二酸二癸酯( didecyl adipate,DDA)、细胞外K+、毒胡萝卜素、细胞外Ca2、U73122及马兜铃酸钠对细胞外液中缘细胞ATP释放的影响.结果 随着巴佛洛霉素A1浓度的增加,细胞外液中ATP的浓度明显下降;当DDA浓度增加时,细胞外液中ATP的浓度几乎呈线性增加.随着细胞外液中的K+浓度的增高,缘细胞释放的ATP浓度呈现上升趋势,当细胞外液中的K+浓度为9.15 mmol/L时,ATP的释放量达到峰值,之后随着K+浓度的继续升高ATP的释放量呈下降趋势.随着毒胡萝卜素浓度的增加,缘细胞释放的ATP浓度呈现明显下降的趋势.当细胞外Ca2+浓度为0 mmol/L时,缘细胞仍然释放ATP;Ca2+浓度增加与ATP的释放呈负相关,但当细胞外的Ca2+浓度达到1.25 mmol/L以上时,ATP的释放量维持在一个较稳定的水平.U73122的浓度在0.25~1.25 μmol/L时,其与缘细胞释放的ATP浓度呈负相关.当马兜铃酸钠的浓度为12.5 ~ 100.0 μmol/L,缘细胞ATP释放呈明显下降的趋势;当其浓度>100.0 μmol/L时,ATP释放浓度趋于平稳.结论 体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,其释放量与钙泵、K+通道状态以及细胞内信号传导通路相关酶的活性有关.%Objective To further confirm release of adenosine triphosphate (ATP) from cultured marginal cells in vitro of stria vascularis in neonatal rat,and to explore the mechanism of ATP release from marginal cells.Methods Isolation and in vitro culture of marginal cells of neonatal rats' cochlea.ATP released by marginal cells in extracellular fluid were detected using bioluminescence assay when add regants separately as follow:bafilomycin At,didecyl adipate (DDA),extracellular K+,thapsigargin,extracellular

  20. Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP.

    Science.gov (United States)

    Roberts, Jonathan A; Evans, Richard J

    2006-02-01

    The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.

  1. Intrarenal localization of the plasma membrane ATP channel pannexin1.

    Science.gov (United States)

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T X; Yu, Alan; Peti-Peterdi, János

    2012-11-15

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

  2. Properties of mutants of haemophilus influenzae deficient in ATP-dependent deoxyribonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.

    1976-01-01

    Eight isogenic Haemophilus influenzae strains whose extracts lack ATP-dependent deoxyribonuclease activity (Add/sup -/ mutants) form three complementation and genetic linkage groups. Since there are known to be three subunits of the enzyme, these data suggest that each of the three genes specifies a different subunit. Gel electrophoresis of partially purified mutant extracts indicates that the smallest subunit is missing in one of the groups but is present in all the other mutants. The mutants are more sensitive to a variety of chemical agents than the wild type. The most sensitive mutants lack the ATPase activity associated with the enzyme. These strains exhibit aberrant incorporation of tritiated thymidine, which starts up more rapidly and shuts off sooner than in the wild type. An extracellular compound is responsible for most of this effect, in that wild type cells put into medium in which Add/sup -/ cells have been growing show a similar aberrant incorporation. The effect of these media can be mimicked by cyclic AMP and cyclic GMP, although millimolar concentrations are required. It is postulated that the Add/sup -/ mutants are more permeable to many substances than the wild type, partly because of the extracellular compound usually surrounding them, and the increased permeability might be responsible for the mutants' nonviability.

  3. Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

    Science.gov (United States)

    Frieden, C; Patane, K

    1985-07-16

    The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.

  4. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    Directory of Open Access Journals (Sweden)

    Jun Adachi

    2015-12-01

    Full Text Available Interactions between ATP and ATP-binding proteins (ATPome are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014 [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014 [1] have been deposited to the ProteomeXchange with identifier PXD001200.

  5. Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Chun Huang

    2015-11-01

    Full Text Available Background/Aims: Adenosine diphosphate ribose (ADPR, a product of β-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs; however the physiological function of extracellular ADPR is unclear. Methods: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y subtypes were examined in pulmonary arteries. Results: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. Conclusion: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

  6. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    Science.gov (United States)

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

  7. Inhibition of ATP-induced calcium influx in HT4 cells by glucocorticoids: involvement of protein kinase A

    Institute of Scientific and Technical Information of China (English)

    Jian-zhong HAN; Wen LIN; Yi-zhang CHEN

    2005-01-01

    Aim: In our previous observations, adenosine triphosphate (ATP) was found to evoke immediate elevations in intracellular free calcium concentration ([Ca2+]i) in HT4 neuroblastoma cells of mice. We tried to see if a brief pretreatment of glucocorticoids could inhibit the Ca2+ response and reveal the underlying signal ing mechanism. Methods: Measurement of [Ca2+]i was carried out using the dual-wavelength fluorescence method with Fura-2 as the indicator. Results: Pre incubation of HT4 cells for 5 min with corticosterone (B) or bovine serum albumin conjugated corticosterone (B-BSA) inhibited the peak [Ca2+]i increments in a concentration-dependent manner. Cortisol and dexamethasone had a similar action, while deoxycorticosterone and cholesterol were ineffective. Both extracellular Ca2+ influx and internal Ca2+ release contributed to ATP-induced [Ca2+]i elevation. The brief treatment with only B attenuated Ca2+ influx. Furthermore, the [Ca2+]i elevation induced by the P2X receptor agonist adenosine 5'-(β,γ-methylene) triphosphate (β,γ-meATP) was also suppressed. The rapid inhibitory effect of B can be reproduced by forskolin 1 mmol/L and blocked by H89 20 mmol/L. Neither nuclear glucocorticoid receptor antagonist mifepristone nor protein kinase C in hibitors influenced the rapid action of B. Conclusion: Our results suggest that glucocorticoids modulate P2X receptor-medicated Ca2+ influx through a membrane-initiated, non-genomic and PKA-dependent pathway in HT4 cells.

  8. Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

    Science.gov (United States)

    Palygin, Oleg; Levchenko, Vladislav; Evans, Louise C.; Blass, Gregory; Cowley, Allen W.; Staruschenko, Alexander

    2015-01-01

    Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made  possible the measuring of cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor are coated with a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs. PMID:26485400

  9. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro.

    Science.gov (United States)

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Zhao, Na; Zhen, Linqing; Fu, Jieli; Yang, Qiangzhen

    2016-09-01

    Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism.

  10. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Directory of Open Access Journals (Sweden)

    Zulfiqar Ahmad

    Full Text Available We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  11. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Science.gov (United States)

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  12. Analysis of extracellular RNA by digital PCR

    Directory of Open Access Journals (Sweden)

    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  13. Extracellular Molecules Involved in Cancer Cell Invasion

    Energy Technology Data Exchange (ETDEWEB)

    Stivarou, Theodora; Patsavoudi, Evangelia, E-mail: epatsavoudi@pasteur.gr [Department of Biochemistry, Hellenic Pasteur Institute, Athens 11521 (Greece); Technological Educational Institute of Athens, Egaleo, Athens 12210 (Greece)

    2015-01-26

    Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

  14. Extracellular Molecules Involved in Cancer Cell Invasion

    Directory of Open Access Journals (Sweden)

    Theodora Stivarou

    2015-01-01

    Full Text Available Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

  15. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    Science.gov (United States)

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  16. Follow the ATP: tumor energy production: a perspective.

    Science.gov (United States)

    Oronsky, Bryan T; Oronsky, Neil; Fanger, Gary R; Parker, Christopher W; Caroen, Scott Z; Lybeck, Michelle; Scicinski, Jan J

    2014-01-01

    As early as the 1920s, the eminent physician and chemist, Otto Warburg, nominated for a second Nobel Prize for his work on fermentation, observed that the core metabolic signature of cancer cells is a high glycolytic flux. Warburg averred that the prime mover of cancer is defective mitochondrial respiration, which drives a switch to an alternative energy source, aerobic glycolysis in lieu of Oxidative Phosphorylation (OXPHOS), in an attempt to maintain cellular viability and support critical macromolecular needs. The cell, deprived of mitochondrial ATP production, must reprogram its metabolism as a secondary survival mechanism to maintain sufficient ATP and NADH levels for macromolecule production, membrane integrity and DNA synthesis as well as maintenance of membrane ionic gradients. A time-tested method to identify and disrupt criminal activity is to "follow the money" since the illicit proceeds from crime are required to underwrite it. By analogy, strategies to target cancer involve following and disrupting the flow of ATP and NADH, the energetic and redox "currencies" of the cell, respectively, since the tumor requires high levels of ATP and NADH, not only for metastasis and proliferation, but also, on a more basic level, for survival. Accordingly, four broad ATP reduction strategies to impact and potentially derail cancer energy production are highlighted herein: 1) small molecule energy-restriction mimetic agents (ERMAs) that target various aspects of energy metabolism, 2) reduction of energy 'subsidization' with autophagy inhibitors, 3) acceleration of ATP turnover to increase energy inefficiency, and 4) dietary energy restriction to limit the energy supply.

  17. ATP formation and ATP hydrolysis during fatiguing, intermittent stimulation of different types of single muscle fibres from Xenopus laevis.

    Science.gov (United States)

    Nagesser, A S; Van der Laarse, W J; Elzinga, G

    1993-12-01

    This report describes changes of the rate of ATP hydrolysis in single, intact muscle fibres during the development of fatigue induced by intermittent tetanic stimulation. High (type 3) and low (type 1) oxidative muscle fibres dissected from the iliofibularis muscle of Xenopus laevis were studied at 20 degrees C. The rate of ATP hydrolysis was calculated during different time intervals from changes in the content of nucleotides, creatine compounds and lactate, as well as lactate efflux and oxygen uptake. During the first phase of intermittent stimulation, phosphocreatine is fully reduced while the rate of oxygen consumption increases to its maximum, the lactate content increases to a maximum level, and a small amount of IMP is formed; the rate of ATP hydrolysis in type 3 fibres is constant while force decreases, whereas the rate decreases approximately in proportion to force in type 1 fibres. After the first phase, the rate of ATP hydrolysis in type 3 fibres decreases slightly and the fibres reach a steady metabolic state in which the rates of ATP formation and hydrolysis are equal; in type 1 fibres a drastic change of the rate of ATP hydrolysis occurs and a steady metabolic state is not reached. On the basis of the time courses of the metabolic changes, it is concluded that the rate of ATP hydrolysis in type 3 fibres is reduced by acidification and/or a reduced calcium efflux from the sarcoplasmic reticulum, whereas in type 1 fibres inorganic phosphate and/or acidification inhibit the rate initially and ADP is a likely candidate to explain the drastic fall of the rate of ATP hydrolysis during late phases of fatiguing stimulation.

  18. Pravastatin-induced improvement in coronary reactivity and circulating ATP and ADP levels in young adults with type 1 diabetes

    Directory of Open Access Journals (Sweden)

    Tuomas Oskari Kiviniemi

    2012-08-01

    Full Text Available Aims: Extracellular ATP and ADP regulate diverse inflammatory, prothrombotic and vasoactive responses in the vasculature. Statins have been shown to modulate their signaling pathways in vitro. We hypothesized that altered intravascular nucleotide turnover modulates vasodilation in patients with type 1 diabetes (T1DM, and this can be partly restored with pravastatin therapy. Methods: In this randomized double blind study, plasma ATP and ADP levels and echocardiography-derived coronary flow velocity response to cold pressor test (CPT were concurrently assessed in 42 normocholesterolemic patients with T1DM (age 30±6 years, LDL cholesterol 2.5±0.6 mmol/L before and after four-month treatment with pravastatin 40 mg/day or placebo (n=22 and n=20, respectively, and in 41 healthy control subjects. Results: Compared to controls, T1DM patients had significantly higher concentrations of ATP (p<0.01 and ADP (p<0.01 and these levels were partly restored after treatment with pravastatin (p=0.002 and p=0.007, respectively, but not after placebo (p=0.06 and p=0.14, respectively. Coronary flow velocity acceleration was significantly lower in T1DM patients compared to control subjects, and it increased from pre- to post-intervention in the pravastatin (p=0.02, but not in placebo group (p=0.15. Conclusions: Pravastatin treatment significantly reduces circulating ATP and ADP levels of T1DM patients, and concurrently improves coronary flow response to CPT. This study provides a novel insight in purinergic mechanisms involved in pleiotropic effects of pravastatin.

  19. ATP and sulfonylurea sensitivity of mutant ATP-sensitive K+ channels in neonatal diabetes: implications for pharmacogenomic therapy.

    Science.gov (United States)

    Koster, Joseph C; Remedi, Maria S; Dao, Crystal; Nichols, Colin G

    2005-09-01

    The prediction that overactivity of the pancreatic ATP-sensitive K(+) channel (K(ATP) channel) underlies reduced insulin secretion and causes a diabetic phenotype in humans has recently been borne out by genetic studies implicating "activating" mutations in the Kir6.2 subunit of K(ATP) as causal in both permanent and transient neonatal diabetes. Here we characterize the channel properties of Kir6.2 mutations that underlie transient neonatal diabetes (I182V) or more severe forms of permanent neonatal diabetes (V59M, Q52R, and I296L). In all cases, the mutations result in a significant decrease in sensitivity to inhibitory ATP, which correlates with channel "overactivity" in intact cells. Mutations can be separated into those that directly affect ATP affinity (I182V) and those that stabilize the open conformation of the channel and indirectly reduce ATP sensitivity (V59M, Q52R, and I296L). With respect to the latter group, alterations in channel gating are also reflected in a functional "uncoupling" of sulfonylurea (SU) block: SU sensitivity of I182V is similar to that of wild-type mutants, but the SU sensitivity of all gating mutants is reduced, with the I296L mutant being resistant to block by tolbutamide (

  20. Extracellular DNA in oral microbial biofilms.

    Science.gov (United States)

    Jakubovics, Nicholas S; Burgess, J Grant

    2015-07-01

    The extracellular matrix of microbial biofilms is critical for surface adhesion and nutrient homeostasis. Evidence is accumulating that extracellular DNA plays a number of important roles in biofilm integrity and formation on hard and soft tissues in the oral cavity. Here, we summarise recent developments in the field and consider the potential of targeting DNA for oral biofilm control.

  1. Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys.

    Science.gov (United States)

    Schwiebert, Erik M; Wallace, Darren P; Braunstein, Gavin M; King, Sandi R; Peti-Peterdi, Janos; Hanaoka, Kazushige; Guggino, William B; Guay-Woodford, Lisa M; Bell, P Darwin; Sullivan, Lawrence P; Grantham, Jared J; Taylor, Amanda L

    2002-04-01

    ATP and its metabolites are potent autocrine agonists that act extracellularly within tissues to affect epithelial function. In polycystic kidneys, renal tubules become dilated and/or encapsulated as cysts, creating abnormal microenvironments for autocrine signaling. Previously, our laboratory has shown that high-nanomolar to micromolar quantities of ATP are released from cell monolayers in vitro and detectable in cyst fluids from microdissected human autosomal dominant polycystic kidney (ADPKD) cysts. Here, we show enhanced ATP release from autosomal recessive polycystic kidney (ARPKD) and ADPKD epithelial cell models. RT-PCR and immunoblotting for P2Y G protein-coupled receptors and P2X purinergic receptor channels show expression of mRNA and/or protein for multiple subtypes from both families. Assays of cytosolic Ca(2+) concentration and secretory Cl(-) transport show P2Y and P2X purinergic receptor-mediated stimulation of Cl(-) secretion via cytosolic Ca(2+)-dependent signaling. Therefore, we hypothesize that autocrine purinergic signaling may augment detrimentally cyst volume expansion in ADPKD or tubule dilation in ARPKD, accelerating disease progression.

  2. A1Ao-ATP synthase of Methanobrevibacter ruminantium couples sodium ions for ATP synthesis under physiological conditions.

    Science.gov (United States)

    McMillan, Duncan G G; Ferguson, Scott A; Dey, Debjit; Schröder, Katja; Aung, Htin Lin; Carbone, Vincenzo; Attwood, Graeme T; Ronimus, Ron S; Meier, Thomas; Janssen, Peter H; Cook, Gregory M

    2011-11-18

    An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A(1)A(o)-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A(1)A(o)-ATP synthase (MbbrA(1)A(o)) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA(1)A(o) was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na(+)-binding signature made up of identical amino acid residues. The purified MbbrA(1)A(o) was stimulated by sodium ions, and Na(+) provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A(1)A(o)-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions.

  3. A carbon nanotubes based ATP apta-sensing platform and its application in cellular assay.

    Science.gov (United States)

    Zhang, Libing; Wei, Hui; Li, Jing; Li, Tao; Li, Dan; Li, Yunhui; Wang, Erkang

    2010-04-15

    In this paper, a sensitive and selective fluorescent aptasensor for adenosine triphosphate (ATP) detection is constructed, based on the noncovalent assembly of dye-labeled ATP aptamer and single-walled carbon nanotubes (SWNTs). In the absence of ATP, the dye tethered to the ATP aptamer is close to SWNTs, which can effectively quench fluorescence of the dye. Upon adding ATP, the fluorophore keeps away from the quencher, since ATP specifically binds to the aptamer and competes with carbon nanotubes, resulting in an increase in the fluorescence intensity. This enables ATP to be detected down to 4.5nM. To the best of our knowledge, this is the most sensitive fluorescent ATP aptasensor. In addition, prominent fluorescence signals were obtained in cellular ATP assays, thus the aptasensor could be used to detect ATP in real samples.

  4. Hyperosmolar sodium chloride is toxic to cultured neurons and causes reduction of glucose metabolism and ATP levels, an increase in glutamate uptake, and a reduction in cytosolic calcium.

    Science.gov (United States)

    Morland, Cecilie; Pettersen, Mi Nguyen; Hassel, Bjørnar

    2016-05-01

    Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10-100mmol/L, for 20h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na(+), since substituting excess Na(+) with choline reduced cell death to control levels, whereas gluconate instead of excess Cl(-) did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na(+)] caused reduction in intracellular free [Ca(2+)], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca(2+)] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia.

  5. Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP/PKA Pathway.

    Science.gov (United States)

    Wang, Chao; Liu, Dandan; Zhang, Cuimiao; Sun, Jiadong; Feng, Weipei; Liang, Xing-Jie; Wang, Shuxiang; Zhang, Jinchao

    2016-05-11

    Novel defect-related hydroxyapatite (DHAP), which combines the advantages of HAP and defect-related luminescence, has the potential application in tissue engineering and biomedical area, because of its excellent capability of monitoring the osteogenic differentiation and material biodegradation. Although the extracellular mechanism of DHAP minerals and PO4(3-) functioning in osteogenic differentiation has been widely studied, the intracellular molecular mechanism through which PO4(3-) mediates osteogenesis of bone mesenchymal stem cells (BMSCs) is not clear. We examined a previously unknown molecular mechanism through which PO4(3-) promoted osteogenesis of BMSCs with an emphasis on adenosine-triphosphate (ATP)-induced cAMP/PKA pathway. Our studies showed that DHAP could be uptaken into lysosome, in which PO4(3-) was released from DHAP, because of the acid environment of lysosome. The released PO4(3-) interacted with ADP to form ATP, and then degraded into adenosine, an ATP metabolite, which interacted with A2b adenosine receptor to activate the cAMP/PKA pathway, resulting in the high expression of osteogenesis-related genes, such as Runx2, BMP-2, and OCN. These findings first revealed the function of ATP-metabolism in bone physiological homeostasis, which may be developed to cure bone metabolic diseases.

  6. Phenomenological analysis of ATP dependence of motor proteins.

    Directory of Open Access Journals (Sweden)

    Yunxin Zhang

    Full Text Available In this study, through phenomenological comparison of the velocity-force data of processive motor proteins, including conventional kinesin, cytoplasmic dynein and myosin V, I found that, the ratio between motor velocities of two different ATP concentrations is almost invariant for any substall, superstall or negative external loads. Therefore, the velocity of motors can be well approximated by a Michaelis-Menten like formula V = [ATP]k(FL([ATP] + K(M, with L the step size, and k(F the external load F dependent rate of one mechanochemical cycle of motor motion in saturated ATP solution. The difference of Michaelis-Menten constant K(M for substall, superstall and negative external load indicates, the configurations at which ATP molecule can bind to motor heads for these three cases might be different, though the expression of k(F as a function of F might be unchanged for any external load F. Verifications of this Michaelis-Menten like formula has also been done by fitting to the recent experimental data.

  7. Phenomenological analysis of ATP dependence of motor protein

    CERN Document Server

    Zhang, Yunxin

    2011-01-01

    In this study, through phenomenological comparison of the velocity-force data of processive motor proteins, including conventional kinesin, cytoplasmic dynein and myosin V, we found that, the ratio between motor velocities of two different ATP concentrations is almost invariant for any substall, superstall or negative external loads. Therefore, the velocity of motor can be well approximated by a Michaelis-Menten like formula $V=\\atp k(F)L/(\\atp +K_M)$, with $L$ the step size, and $k(F)$ the external load $F$ dependent rate of one mechanochemical cycle of motor motion in saturated ATP solution. The difference of Michaelis-Menten constant $K_M$ for substall, superstall and negative external load indicates, the ATP molecule affinity of motor head for these three cases are different, though the expression of $k(F)$ as a function of $F$ might be unchanged for any external load $F$. Verifications of this Michaelis-Menten like formula has also been done by fitting to the recent experimental data.

  8. Rotation and structure of FoF1-ATP synthase.

    Science.gov (United States)

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

  9. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Fumin; Telesco, Shannon E.; Liu, Yingting; Radhakrishnan, Ravi; Lemmon, Mark A. (UPENN); (UPENN-MED)

    2010-06-21

    ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues - including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region - although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K{sub d} of approximately 1.1 {micro}M. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened {alpha}C-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the 'inactive-like'configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.

  10. P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

    Science.gov (United States)

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2011-04-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

  11. Extracellular DNA metabolism in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Scott eChimileski

    2014-02-01

    Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

  12. Extracellular Nucleotides in Exercise: Possible Effect on Brain Metabolism.

    Science.gov (United States)

    Forrester, Tom

    1979-01-01

    A review of experiments which demonstrate the release of ATP from skeletal muscle, cardiac muscle, and active brain tissue. Effects of exogenously applied ATP to brain tissue are discussed in relation to whole body exercise. (Author/SA)

  13. Preservative efficacy screening of pharmaceutical formulations using ATP bioluminescence.

    Science.gov (United States)

    Kramer, Mateja; Suklje-Debeljak, Helena; Kmetec, Vojko

    2008-05-01

    The preservative challenge test is a method used to determine the efficacy of a preservation system in a pharmaceutical or cosmetic formulation. However, such testing is a labor-intensive, repetitive task often requiring days before results can be generated. Several alternatives to traditional colony-count techniques have been developed. A study using pure suspensions of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Candida albicans, and Aspergillus niger showed that the accuracy, repeatability, and linearity of the Pallchek luminometer ATP bioluminescence (ATP-B) system was equivalent to the traditional colony-count method. In any case, the method proved sensitive enough to follow the effect of preservatives on a number of test microorganisms, indicating the applicability of the ATP-B method for preservative screening studies in various pharmaceutical formulations.

  14. Hypophosphatemia promotes lower rates of muscle ATP synthesis

    DEFF Research Database (Denmark)

    Pesta, Dominik H; Tsirigotis, Dimitrios N; Befroy, Douglas E

    2016-01-01

    Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [(31)P]-magnetic resonance spectroscopy....... By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in NaPi2a knockout mice (NaPi2a(-/-)) compared with their wild-type littermate controls...... hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum Pi content and muscle VATP After oral Pi repletion and normalization of serum Pi levels, muscle VATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP...

  15. ATP Production in Chlamydomonas reinhardtii Flagella by Glycolytic Enzymes

    DEFF Research Database (Denmark)

    Mitchell, Beth F; Pedersen, Lotte B; Feely, Michael

    2005-01-01

    Eukaryotic cilia and flagella are long, thin organelles, and diffusion from the cytoplasm may not be able to support the high ATP concentrations needed for dynein motor activity. We discovered enzyme activities in the Chlamydomonas reinhardtii flagellum that catalyze three steps of the lower half...... of glycolysis (phosphoglycerate mutase, enolase, and pyruvate kinase). These enzymes can generate one ATP molecule for every substrate molecule consumed. Flagellar fractionation shows that enolase is at least partially associated with the axoneme, whereas phosphoglycerate mutase and pyruvate kinase primarily...... reside in the detergent-soluble (membrane + matrix) compartments. We further show that axonemal enolase is a subunit of the CPC1 central pair complex and that reduced flagellar enolase levels in the cpc1 mutant correlate with the reduced flagellar ATP concentrations and reduced in vivo beat frequencies...

  16. ATP economy of force maintenance in human tibialis anterior muscle

    DEFF Research Database (Denmark)

    Nakagawa, Yoshinao; Ratkevicius, Aivaras; Mizuno, Masao

    2005-01-01

    PURPOSE: The aim of this study was investigate ATP economy of force maintenance in the human tibialis anterior muscle during 60 s of anaerobic voluntary contraction at 50% of maximum voluntary contraction (MVC). METHODS: ATP turnover rate was evaluated using P magnetic resonance spectroscopy (P......-MRS). The total volume of ankle dorsiflexor muscles was assessed by H magnetic resonance imaging (MRI) (H-MRI), and the fiber type composition of the tibialis anterior muscle was evaluated using histochemical analysis of muscle biopsies. RESULTS: The tibialis anterior muscle occupied 59.7 +/- 0.6% (mean +/- SEM......) of the total ankle dorsiflexor muscle volume, which was 267 +/- 10 cm. Relative cross-sectional areas occupied by Type I, IIA, and IIB fibers in the tibialis anterior were 69.3 +/- 2.2, 27.4 +/- 2.76, and 3.2 +/- 1.0%, respectively. ATP economy of force maintenance did not change significantly during the 60-s...

  17. The Arabidopsis thaliana lectin receptor kinase LecRK-I.9 is required for full resistance to Pseudomonas syringae and affects jasmonate signalling.

    Science.gov (United States)

    Balagué, Claudine; Gouget, Anne; Bouchez, Olivier; Souriac, Camille; Haget, Nathalie; Boutet-Mercey, Stéphanie; Govers, Francine; Roby, Dominique; Canut, Hervé

    2016-07-11

    On microbial attack, plants can detect invaders and activate plant innate immunity. For the detection of pathogen molecules or cell wall damage, plants employ receptors that trigger the activation of defence responses. Cell surface proteins that belong to large families of lectin receptor kinases are candidates to function as immune receptors. Here, the function of LecRK-I.9 (At5g60300), a legume-type lectin receptor kinase involved in cell wall-plasma membrane contacts and in extracellular ATP (eATP) perception, was studied through biochemical, gene expression and reverse genetics approaches. In Arabidopsis thaliana, LecRK-I.9 expression is rapidly, highly and locally induced on inoculation with avirulent strains of Pseudomonas syringae pv. tomato (Pst). Two allelic lecrk-I.9 knock-out mutants showed decreased resistance to Pst. Conversely, over-expression of LecRK-I.9 led to increased resistance to Pst. The analysis of defence gene expression suggests an alteration of both the salicylic acid (SA) and jasmonic acid (JA) signalling pathways. In particular, LecRK-I.9 expression during plant-pathogen interaction was dependent on COI1 (CORONATINE INSENSITIVE 1) and JAR1 (JASMONATE RESISTANT 1) components, and JA-responsive transcription factors (TFs) showed altered levels of expression in plants over-expressing LecRK-I.9. A similar misregulation of these TFs was obtained by JA treatment. This study identified LecRK-I.9 as necessary for full resistance to Pst and demonstrated its involvement in the control of defence against pathogens through a regulation of JA signalling components. The role of LecRK-I.9 is discussed with regard to the potential molecular mechanisms linking JA signalling to cell wall damage and/or eATP perception.

  18. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

    OpenAIRE

    2010-01-01

    Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphib...

  19. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    OpenAIRE

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control...

  20. Functional studies of ATP sulfurylase from Penicillium chrysogenum

    Energy Technology Data Exchange (ETDEWEB)

    Seubert, P.A.

    1985-01-01

    ATP sulfurylase from Penicillium chrysogenum has a specific activity (V/sub max/) of 6-7 units x mg protein/sup -1/ determined with the physiological substrates of MgATP and SO/sub 4//sup 2 -/ and assayed by (A) initial velocity measurements with APS kinase and inorganic pyrophosphatase present and (B) analysis of nonlinear reaction progress curves. The fact both assays give the same results show the intrinsic activity of ATP sulfurylase is much higher than previously reported. In initial velocity dead-end inhibition studies, the sulfate analog S/sub 2/O/sub 3//sup 2 -/ is a competitive inhibitor of SO/sub 42/..sqrt.. and a noncompetitive inhibitor of MgATP. Monovalent oxyanions such as NO/sub 3//sup -/, ClO/sub 3//sup -/, ClO/sub 4//sup -/, and FSO/sub 3//sup -/ behave as uncompetitive inhibitors of MgATP and thus seem not to be true sulfate analogs. The reverse reaction was assayed by the pyrophosphate dependent release of /sup 35/SO/sub 4//sup 2 -/ from AP/sup 35/S. Product inhibition by MgATP or SO/sub 4//sup 2 -/ is competitive with APS and mixed-type with PP/sub i/. Imidodiphosphate can serve as an alternative substrate for PP/sub i/. ATP sulfurylase binds (but does not hydrolyze) APS. A Scatchard plot of the APS binding is nonlinear, suggesting at least two types of sites. The cumulative results are qualitatively consistent with the random addition of MgATP and SO/sub 4//sup 2 -/ and the ordered release of first MgPP/sub i/ then APS, with APS release being partially rate limiting. Certain quantitative discrepancies suggest either an unknown variable (e.g. enzyme concentration) complicates the analysis or, in light of binding studies that the actual mechanism is more complicated (e.g. alternating sites) than any of the conventional models examined.

  1. ATP-promoted amyloidosis of an amyloid beta peptide.

    Science.gov (United States)

    Exley, C

    1997-10-20

    Amyloidosis is implicated in the aetiology of a number of disorders of human health. The factors that influence its instigation and subsequent rate of progress are the subject of a considerable research effort. The peptide fragment A beta(25-35) is amyloidogenic and has proven to be a useful model of the processes involved in amyloidosis. It is demonstrated herein that the assembly of A beta(25-35) into thioflavin T-reactive fibrils and their subsequent rearrangement into advanced glycation endproducts is accelerated by ATP. Aluminium potentiated these effects of ATP, suggesting a possible link with the aetiology of amyloidoses in vivo.

  2. Th1/Th17 cell induction and corresponding reduction in ATP consumption following vaccination with the novel Mycobacterium tuberculosis vaccine MVA85A.

    Science.gov (United States)

    Griffiths, Kristin L; Pathan, Ansar A; Minassian, Angela M; Sander, Clare R; Beveridge, Natalie E R; Hill, Adrian V S; Fletcher, Helen A; McShane, Helen

    2011-01-01

    Vaccination with Bacille Calmette-Guérin (BCG) has traditionally been used for protection against disease caused by the bacterium Mycobacterium tuberculosis (M.tb). The efficacy of BCG, especially against pulmonary tuberculosis (TB) is variable. The best protection is conferred in temperate climates and there is close to zero protection in many tropical areas with a high prevalence of both tuberculous and non-tuberculous mycobacterial species. Although interferon (IFN)-γ is known to be important in protection against TB disease, data is emerging on a possible role for interleukin (IL)-17 as a key cytokine in both murine and bovine TB vaccine studies, as well as in humans. Modified Vaccinia virus Ankara expressing Antigen 85A (MVA85A) is a novel TB vaccine designed to enhance responses induced by BCG. Antigen-specific IFN-γ production has already been shown to peak one week post-MVA85A vaccination, and an inverse relationship between IL-17-producing cells and regulatory T cells expressing the ectonucleosidease CD39, which metabolises pro-inflammatory extracellular ATP has previously been described. This paper explores this relationship and finds that consumption of extracellular ATP by peripheral blood mononuclear cells from MVA85A-vaccinated subjects drops two weeks post-vaccination, corresponding to a drop in the percentage of a regulatory T cell subset expressing the ectonucleosidase CD39. Also at this time point, we report a peak in co-production of IL-17 and IFN-γ by CD4(+) T cells. These results suggest a relationship between extracellular ATP and effector responses and unveil a possible pathway that could be targeted during vaccine design.

  3. Th1/Th17 cell induction and corresponding reduction in ATP consumption following vaccination with the novel Mycobacterium tuberculosis vaccine MVA85A.

    Directory of Open Access Journals (Sweden)

    Kristin L Griffiths

    Full Text Available Vaccination with Bacille Calmette-Guérin (BCG has traditionally been used for protection against disease caused by the bacterium Mycobacterium tuberculosis (M.tb. The efficacy of BCG, especially against pulmonary tuberculosis (TB is variable. The best protection is conferred in temperate climates and there is close to zero protection in many tropical areas with a high prevalence of both tuberculous and non-tuberculous mycobacterial species. Although interferon (IFN-γ is known to be important in protection against TB disease, data is emerging on a possible role for interleukin (IL-17 as a key cytokine in both murine and bovine TB vaccine studies, as well as in humans. Modified Vaccinia virus Ankara expressing Antigen 85A (MVA85A is a novel TB vaccine designed to enhance responses induced by BCG. Antigen-specific IFN-γ production has already been shown to peak one week post-MVA85A vaccination, and an inverse relationship between IL-17-producing cells and regulatory T cells expressing the ectonucleosidease CD39, which metabolises pro-inflammatory extracellular ATP has previously been described. This paper explores this relationship and finds that consumption of extracellular ATP by peripheral blood mononuclear cells from MVA85A-vaccinated subjects drops two weeks post-vaccination, corresponding to a drop in the percentage of a regulatory T cell subset expressing the ectonucleosidase CD39. Also at this time point, we report a peak in co-production of IL-17 and IFN-γ by CD4(+ T cells. These results suggest a relationship between extracellular ATP and effector responses and unveil a possible pathway that could be targeted during vaccine design.

  4. and extracellular laccase isoenzymes from Pleurotus ostreatus ...

    African Journals Online (AJOL)

    ZMG

    Colonia Vicentina, Delegación Iztapalapa, 09340 México D.F., México. ... In this study, extracellular laccase enzymes produced by Pleurotus ostreatus was identified in .... the intracellular forms), through the modified zymography method of.

  5. Alternative methods for characterization of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Fatemeh eMomen-Heravi

    2012-09-01

    Full Text Available Extracellular vesicles are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell-cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bioreactive macromolecules such as nucleic acids, proteins and lipids. A major limitation in this emerging new field is the availability/awareness of techniques to isolate and properly characterize Extracellular vesicles. The lack of gold standards makes comparing different studies very difficult and may potentially hinder some Extracellular vesicles -specific evidence. Characterization of Extracellular vesicles has also recently seen many advances with the use of Nanoparticle Tracking Analysis (NTA, flow cytometry, cryo-EM instruments and proteomic technologies. In this review, we discuss the latest developments in translational technologies involving characterization methods including the facts in their support and the challenges they face.

  6. Elucidation of the ATP7B N-domain Mg2+-ATP coordination site and its allosteric regulation.

    Directory of Open Access Journals (Sweden)

    Claude Hercend

    Full Text Available The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain of the Mg(2+-ATP coordination site and answer to the controversial role of the Mg(2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg(2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process.

  7. Bacterial binding to extracellular proteins - in vitro adhesion

    DEFF Research Database (Denmark)

    Schou, C.; Fiehn, N.-E.

    1999-01-01

    Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis......Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis...

  8. The extracellular RNA complement of Escherichia coli.

    Science.gov (United States)

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-21

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. © 2015 The

  9. Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

    Directory of Open Access Journals (Sweden)

    Kimberly L. James

    2016-08-01

    Full Text Available Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1 for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4 in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.

  10. Characterization of a modified ROCK2 protein that allows use of N6-ATP analogs for the identification of novel substrates

    OpenAIRE

    Couzens, Amber L.; Gill, R. Montgomery; Scheid, Michael P.

    2014-01-01

    Background The Rho-associated coiled-coil kinase-2 (ROCK2) is an important signaling transducer in the transmission of extracellular signals effecting organization of the actin cytoskeleton. ROCK2 has been implicated in numerous pathologies and the current focus is on understanding the molecular events that couple ROCK2 activity to biological function. To aid in the search for new ROCK2 substrates, we have developed an analog-sensitive (AS) ROCK2 protein that allows the use of selective ATP a...

  11. Transcriptome of extracellular vesicles released by hepatocytes.

    Directory of Open Access Journals (Sweden)

    Felix Royo

    Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

  12. Structural changes during ATP hydrolysis activity of the ATP synthase from Escherichia coli as revealed by fluorescent probes.

    Science.gov (United States)

    Turina, P

    2000-08-01

    F1F0-ATPase complexes undergo several changes in their tertiary and quaternary structure during their functioning. As a possible way to detect some of these different conformations during their activity, an environment-sensitive fluorescence probe was bound to cysteine residues, introduced by site-directed mutagenesis, in the gamma subunit of the Escherichia coli enzyme. Fluorescence changes and ATP hydrolysis rates were compared under various conditions in F1 and in reconstituted F1F0. The results are discussed in terms of possible modes of operation of the ATP synthases.

  13. 'Domino' systems biology and the 'A' of ATP.

    Science.gov (United States)

    Verma, Malkhey; Zakhartsev, Maksim; Reuss, Matthias; Westerhoff, Hans V

    2013-01-01

    We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  14. Teacher Development Program for ATP 2000. Project Report.

    Science.gov (United States)

    Sutphin, Dean; And Others

    Agri Tech Prep 2000 (ATP 2000) is a 4-year tech prep program linking high school and postsecondary curricula designed to prepare New York students for careers in agriculture or acceptance into a college program in agriculture. Because teacher development was designated an integral project component for fiscal year 1991-1992, a weeklong teacher…

  15. ATP Interior Noise Technology and Flight Demonstration Program

    Science.gov (United States)

    Stephens, David G.; Powell, Clemans A.

    1988-01-01

    The paper provides an overview of the ATP (Advanced Turboprop Program) acoustics program with emphasis on the NASA technology program and the recent NASA/Industry demonstration programs aimed at understanding and controlling passenger cabin noise. Technology developments in propeller (source) noise, cabin noise transmission, and subjective acoustics are described. Finally, an overview of the industry demonstrator programs is presented.

  16. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    Science.gov (United States)

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  17. Interaction between ATP, metal ions, glycine, and several minerals

    Science.gov (United States)

    Rishpon, J.; Ohara, P. J.; Lawless, J. G.; Lahav, N.

    1982-01-01

    Interactions between ATP, glycine and montmorillonite and kaolinite clay minerals in the presence of various metal cations are investigated. The adsorption of adenine nucleotides on clays and Al(OH)3 was measured as a function of pH, and glycine condensation was followed in the presence of ATP, ZnCl2, MgCl2 and either kaolinite or montmorillonite. The amounts of ATP and ADP adsorbed are found to decrease with increasing Ph, and to be considerably enhanced in experiments with Mg(2+)- and Zn(2+)-montmorillonite with respect to Na(+)-montmorillonite. The effects of divalent cations are less marked in kaolinite. Results for Al(OH)3 show the importance of adsorption at clay platelet edges at high pH. The decomposition of ATP during drying at high temperature is observed to be inhibited by small amounts of clay, vacuum, or Mg(2+) or Zn(2+) ions, and to be accompanied by peptide formation in the presence of glycine. Results suggest the importance of Zn(2+) and Mg(2+) in chemical evolution.

  18. Detection of ATP and NADH: A Bioluminescent Experience.

    Science.gov (United States)

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  19. Protection of ATP-Depleted Cells by Impermeant Strychnine Derivatives

    Science.gov (United States)

    Dong, Zheng; Venkatachalam, Manjeri A.; Weinberg, Joel M.; Saikumar, Pothana; Patel, Yogendra

    2001-01-01

    Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine. PMID:11238050

  20. IV ATP potentiates midazolam sedation as assessed by bispectral index.

    Science.gov (United States)

    Sakurai, Satoru; Fukunaga, Atsuo; Ichinohe, Tatsuya; Kaneko, Yuzuru

    2014-01-01

    In this study, by measuring bispectral index (BIS), we tested the hypothesis that intravenous adenosine 5'-triphosphate (ATP) infusion would deepen the level of midazolam-induced sedation. Ten healthy volunteers underwent 2 experiments with at least 2 weeks' interval: immediately after intravenous bolus administration of midazolam (0.04 mg/kg), they received continuous infusion of either ATP infusion (100 μg/kg/min) or placebo (saline) for 40 minutes in a double-blind, randomized, crossover manner. Changes in BIS values and responsiveness to verbal command as well as cardiorespiratory variables were observed throughout the study periods. Administration of midazolam alone reduced BIS value from control: 97 ± 1 to 68 ± 18 at 25 minutes, which was accompanied by significant cardiopulmonary depressant effects, while maintaining responsiveness to verbal command (consciousness) throughout the study period. Coadministration of ATP with midazolam further reduced BIS value to 51 ± 13, associated with complete loss of consciousness without adverse effect on the cardiorespiratory systems. We conclude that the addition of ATP infusion to midazolam significantly enhances midazolam sedation without disturbing cardiorespiratory functions.

  1. Motor pathway excitability in ATP13A2 mutation carriers

    DEFF Research Database (Denmark)

    Zittel, S; Kroeger, J; van der Vegt, J P M

    2012-01-01

    OBJECTIVE: To describe excitability of motor pathways in Kufor-Rakeb syndrome (PARK9), an autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration caused by a mutation in the ATP13A2 gene, using transcranial magnetic stimulation (TMS). METHODS: Five members of a Chilean family...

  2. ATP-enhanced peroxidase-like activity of gold nanoparticles.

    Science.gov (United States)

    Shah, Juhi; Purohit, Rahul; Singh, Ragini; Karakoti, Ajay Singh; Singh, Sanjay

    2015-10-15

    Gold nanoparticles (AuNPs) are known to possess intrinsic biological peroxidase-like activity that has applications in development of numerous biosensors. The reactivity of the Au atoms at the surface of AuNPs is critical to the performance of such biosensors, yet little is known about the effect of biomolecules and ions on the peroxidase-like activity. In this work, the effect of ATP and other biologically relevant molecules and ions over peroxidase-like activity of AuNPs are described. Contrary to the expectation that nanoparticles exposed to biomolecules may lose the catalytic property, ATP and ADP addition enhanced the peroxidase-like activity of AuNPs. The catalytic activity was unaltered by the addition of free phosphate, sulphate and carbonate anions however, addition of ascorbic acid to the reaction mixture diminished the intrinsic peroxidase-like activity of AuNPs, even in the presence of ATP and ADP. In contrast to AuNPs, ATP did not synergize and improve the peroxidase activity of the natural peroxidase enzyme, horseradish peroxidase.

  3. Familial Hemiplegic Migraine With ATP1A2 Mutations

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2007-05-01

    Full Text Available Three children with prolonged hemiplegia following severe unilateral headache and having mutations in ATP1A2 are reported from UCLA School of Medicine, Los Angeles, CA; University Children’s Hospital, Zurich, Switzerland; and Wake Forest University School of Medicine, Winston-Salem, NC.

  4. Misconceptions on ATP thermodynamic role in cellular processes

    Directory of Open Access Journals (Sweden)

    R. M. Martins

    2011-04-01

    Full Text Available The occurrence and permanence of misconceptions have negative implications on the learning processes  since it impairs theconstruction of significant learning. Misconceptions correction is a complex task due to the difficulties intheir detection and high resistance to their removal. The mainobjective of the present work was to investigate misconceptions about the thermodynamic role of theATP in cellular processes. Tests were realized with high school (HS, undergraduate (UG and graduate students involved in PhD programs (G. In this survey students answered a 15 item questionnaire dealing with the ATP role as the cellular energy source. The stability of such misconceptionswere verified: one result shows that 68% HS, 92% UG and 91% G students state that the energy from ATP hydrolysis is responsiblefor driving cellular processes. Overall results show that students carry misconceptions on basic thermodynamic concepts such as energy transfer and chemical reactions spontaneity. One source of the prevalence of the discussed misconceptions aretextbooks, where schemes, figures and even text early introducefalse concepts on the ATP role.

  5. Carbon and energy metabolism of atp mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole

    1992-01-01

    . The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production...

  6. ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

    Science.gov (United States)

    Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D.; Ritz, Thorsten; Brettel, Klaus

    2014-06-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH. radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD.-, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396-. Its negative charge could trigger conformational changes necessary for signal transduction.

  7. Dual functions of a monoclonal antibody against cell surface F1F0 ATP synthase on both HUVEC and tumor cells

    Institute of Scientific and Technical Information of China (English)

    Xia ZHANG; Feng GAO; Li-li YU; Yan PENG; Hong-hai LIU; Jin-ying LIU; Ming YIN; Jian NI

    2008-01-01

    Aim: To generate a monoclonal antibody (McAb) against cell surface FI F0 ATP synthase (ATPase) and observe its antitumoral activity on both human umbilical vein endothelial cells (HUVEC) and tumor cells. Methods: Hybridoma cells secret- ing McAb against ATPase were produced by polyethylene glycol-mediated fu- sions and screened by ELISA. The specificity of McAb was demonstrated by immunofluorescence and confocal imaging, as well as flow cytometry analysis. After the blockade of surface ATPase with McAb on HUVEC and human breast adenocarcinoma MDA-MB-231 cells, an ATP determination kit and CellTiter96 Aqueous Assay (MTS) assay were used to detect the effect of the antibody on extracellular ATP modification and cell proliferation. A cellular cytotoxicity assay in combination with doxorubicin, and a cell migration assay on MDA-MB-231 cells were used to determine the antitumoral activity. Finally, a HUVEC tube formation assay was used to detect the antiangiogenic effect of McAb178-5G10. Results: A monoclonal antibody (McAb178-SG10) against the β-subunit of ATPase was generated, and its reactivity toward HUVEC and tumor cells was studied. We demonstrate that McAb178-SG10 binds to ATPase at the cell surface, where it is able to inhibit ATP synthesis. This antibody also prevents the proliferation of HUVEC and MDA-MB-231 cells. Furthermore, McAb 178-5G l0 enhances the tumoricidal effects of doxorubicin (P<0.05), inhibits the migration of MDA-MB- 231 in transwell assays (P<0.01), and disrupts HUVEC tube formation on Matrigel (P<0.01). Conclusion: McAb178-5GI0 binds preferentially to cell surface ATPase, blocks ATP synthesis, and exhibits both antiangiogenic and antitumorigenic effects.

  8. Dissolved inorganic carbon uptake in Thiomicrospira crunogena XCL-2 is Δp- and ATP-sensitive and enhances RubisCO-mediated carbon fixation.

    Science.gov (United States)

    Menning, Kristy J; Menon, Balaraj B; Fox, Gordon; Scott, Kathleen M

    2016-03-01

    The gammaproteobacterium Thiomicrospira crunogena XCL-2 is an aerobic sulfur-oxidizing hydrothermal vent chemolithoautotroph that has a CO2 concentrating mechanism (CCM), which generates intracellular dissolved inorganic carbon (DIC) concentrations much higher than extracellular, thereby providing substrate for carbon fixation at sufficient rate. This CCM presumably requires at least one active DIC transporter to generate the elevated intracellular concentrations of DIC measured in this organism. In this study, the half-saturation constant (K CO2) for purified carboxysomal RubisCO was measured (276 ± 18 µM) which was much greater than the K CO2 of whole cells (1.03 µM), highlighting the degree to which the CCM facilitates CO2 fixation under low CO2 conditions. To clarify the bioenergetics powering active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis (DCCD) or proton potential (CCCP). Incubations with each of these inhibitors resulted in diminished intracellular ATP, DIC, and fixed carbon, despite an absence of an inhibitory effect on proton potential in the DCCD-incubated cells. Electron transport complexes NADH dehydrogenase and the bc 1 complex were found to be insensitive to DCCD, suggesting that ATP synthase was the primary target of DCCD. Given the correlation of DIC uptake to the intracellular ATP concentration, the ABC transporter genes were targeted by qRT-PCR, but were not upregulated under low-DIC conditions. As the T. crunogena genome does not include orthologs of any genes encoding known DIC uptake systems, these data suggest that a novel, yet to be identified, ATP- and proton potential-dependent DIC transporter is active in this bacterium. This transporter serves to facilitate growth by T. crunogena and other Thiomicrospiras in the many habitats where they are found.

  9. Online damage inspection of optics for ATP system

    Science.gov (United States)

    Chen, Jing; Jiang, Yu; Mao, Yao; Gan, Xun; Liu, Qiong

    2016-09-01

    In the Electro-Optical acquisition-tracking-pointing system (ATP), the optical components will be damaged with the several influencing factors. In this situation, the rate will increase sharply when the arrival of damage to some extent. As the complex processing techniques and long processing cycle of optical components, the damage will cause the great increase of the system development cost and cycle. Therefore, it is significant to detect the laser-induced damage in the ATP system. At present, the major research on the on-line damage detection technology of optical components is for the large optical system in the international. The relevant detection systems have complicated structures and many of components, and require enough installation space reserved, which do not apply for ATP system. To solve the problem mentioned before, This paper use a method based on machine vision to detect the damage on-line for the present ATP system. To start with, CCD and PC are used for image acquisition. Secondly, smoothing filters are used to restrain false damage points produced by noise. Then, with the shape feature included in the damage image, the OTSU Method which can define the best segmentation threshold automatically is used to achieve the goal to locate the damage regions. At last, we can supply some opinions for the lifetime of the optical components by analyzing the damage data, such as damage area, damage position. The method has the characteristics of few-detectors and simple-structures which can be installed without any changes of the original light path. With the method, experimental results show that it is stable and effective to achieve the goal of detecting the damage of optical components on-line in the ATP system.

  10. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  11. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-01-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways. PMID:28195155

  12. Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor.

    Directory of Open Access Journals (Sweden)

    Milos B Rokic

    Full Text Available The binding of ATP to trimeric P2X receptors (P2XR causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.

  13. Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9)

    NARCIS (Netherlands)

    Wolters, Justina Clarinda; Abele, Rupert; Tampé, Robert

    2005-01-01

    The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a h

  14. Sensitization by extracellular Ca(2+) of rat P2X(5) receptor and its pharmacological properties compared with rat P2X(1).

    Science.gov (United States)

    Wildman, Scott S; Brown, Sean G; Rahman, Mary; Noel, Carole A; Churchill, Linda; Burnstock, Geoffrey; Unwin, Robert J; King, Brian F

    2002-10-01

    The recombinant rat P2X(5) (rP2X(5)) receptor, a poorly understood ATP-gated ion channel, was studied under voltage-clamp conditions and compared with the better understood homomeric rP2X(1) receptor with which it may coexist in vivo. Expressed in defolliculated Xenopus laevis oocytes, rP2X(5) responded to ATP with slowly desensitizing inward currents that, for successive responses, ran down in the presence of extracellular Ca(2+) (1.8 mM). Replacement of Ca(2+) with either Ba(2+) or Mg(2+) prevented rundown, although agonist responses were very small, whereas reintroduction of Ca(2+) for short periods of time ( 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) > suramin > reactive blue 2 (RB-2) > diinosine pentaphosphate (Ip(5)I). In contrast, the potency order at rP2X(1) was TNP-ATP = Ip(5)I > PPADS > suramin = RB-2. Thus, the Ca(2+)-sensitized homomeric rP2X(5) receptor is similar in agonist profile to homomeric rP2X(1)-although it can be distinguished from the latter by GTP agonism, antagonist profile, and the modulatory effects of H(+) and Zn(2+) ions.

  15. Human beta-defensin-2 and -3 enhance pro-inflammatory cytokine expression induced by TLR ligands via ATP-release in a P2X7R dependent manner.

    Science.gov (United States)

    Wanke, Daniela; Mauch-Mücke, Katrin; Holler, Ernst; Hehlgans, Thomas

    2016-11-01

    Our previous results indicate that HBD2 and HBD3 are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner. In this study we report that pre-stimulation of primary human macrophages or THP-1 cells with HBD2 or HBD3 results in a synergistic, enhanced expression of pro-inflammatory cytokines and chemokines induced by TLR ligand re-stimulation. Experiments using specific inhibitors of the ATP-gated channel receptor P2X7 or its functional ligand ATP, suggest that the enhanced expression of pro-inflammatory cytokines and chemokines seems to be mediated by P2X7R. Furthermore, our data provide evidence that beta-defensins do not directly interact with P2X7R but rather induce the release of intracellular ATP. Interference with ATP release abrogated the synergistic effect mediated by HBD2 and HBD3 pre-stimulation in THP-1 cells. However, extracellular ATP alone seems not to be sufficient to elicit the enhanced synergistic effect on cytokine and chemokine expression observed by pre-stimulation of primary human macrophages or THP-1 cells with HBD2 or HBD3. Collectively, our findings provide new insights into the molecular mechanisms how HBD2 and HBD3 interact with cells of myeloid origin and demonstrate their immuno-modulating functions during innate immune responses.

  16. P2X4 assembles with P2X7 and pannexin-1 in gingival epithelial cells and modulates ATP-induced reactive oxygen species production and inflammasome activation.

    Directory of Open Access Journals (Sweden)

    Shu-Chen Hung

    Full Text Available We have previously reported that Porphyromonas gingivalis infection of gingival epithelial cells (GEC requires an exogenous danger signal such as ATP to activate an inflammasome and caspase-1, thereby inducing secretion of interleukin (IL-1β. Stimulation with extracellular ATP also stimulates production of reactive oxygen species (ROS in GEC. However, the mechanism by which ROS is generated in response to ATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we revealed that the purinergic receptor P2X(4 is assembled with the receptor P2X(7 and its associated pore, pannexin-1. ATP induces ROS production through a complex consisting of the P2X(4, P2X(7, and pannexin-1. P2X(7-mediated ROS production can activate the NLRP3 inflammasome and caspase-1. Furthermore, separate depletion or inhibition of P2X(4, P2X(7, or pannexin-1 complex blocks IL-1β secretion in P. gingivalis-infected GEC following ATP treatment. However, activation via P2X(4 alone induces ROS generation but not inflammasome activation. These results suggest that ROS is generated through stimulation of a P2X(4/P2X(7/pannexin-1 complex, and reveal an unexpected role for P2X(4, which acts as a positive regulator of inflammasome activation during microbial infection.

  17. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    Directory of Open Access Journals (Sweden)

    Jonathon eTelianidis

    2013-08-01

    Full Text Available Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-Type ATPases (copper-ATPases, ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  18. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis.

    Science.gov (United States)

    Telianidis, Jonathon; Hung, Ya Hui; Materia, Stephanie; Fontaine, Sharon La

    2013-01-01

    Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  19. Cell apoptosis and expression of ATP7A in brain of Atp7btx-J mice%Atp7btx-J小鼠脑组织细胞凋亡及ATP7A表达研究

    Institute of Scientific and Technical Information of China (English)

    胡璟; 焦先婷; 刘晓青; 余晓刚; 何振娟; 张拥军

    2013-01-01

    目的 通过对Atp7btx-J小鼠脑组织不同部位细胞凋亡的分析,钙、铜含量的测定,以及铜转运ATP酶ATP7A表达的研究,初步探讨肝豆状核变性致神经系统损伤的作用机制.方法 分离20周龄Atp7btx-J小鼠大脑皮层、小脑、基底神经节及海马区脑组织,Hoechst染色后分析细胞凋亡情况,电感耦合等离子体质谱法测定钙、铜含量,Real-Time PCR检测不同部位ATP7A mRNA 的表达量.结果 与野生型小鼠相比,纯合型小鼠小脑及基底神经节区细胞凋亡最为显著(P<0.05),小脑、基底神经节及海马区的铜和钙含量明显升高(P<0.01),脑组织不同部位ATP7A mRNA表达下调,其中基底神经节和小脑部位ATP7A mRNA表达的差异有统计学意义(P<0.05).结论 肝豆状核变性致神经系统损伤是多因素共同作用的结果,铜在脑内特殊部位的异常蓄积是始动因素,钙介导的细胞凋亡是导致神经系统损伤的重要因素,ATP7A在脑内不能代偿性地协助排出大量蓄积的铜可能加剧了神经系统的损伤.%Objective To explore the mechanism of nervous system injury in Wilson's disease by analysis of cell apoptosis,determination of concentrations of Ca and Cu and detection of expression of ATP7A in different parts of brain tissues of Atp7btx-J mice.Methods The tissues of cerebral cortex,cerebellum,basal ganglia and hippocampus of Atp7btx-J mice aged 20 weeks were isolated,the cell apoptosis was analyzed after Hoechst staining,the concentrations of Ca and Cu were determined by inductively coupled plasma mass spectrometry,and the expression of ATP7A mRNA in different parts was detected by Real-Time PCR.Results Compared with wild type mice,the cell apoptosis in cerebellum and basal ganglia of homozygous mice was most significant (P < 0.05).The concentrations of Ca and Cu in cerebellum,basal ganglia and hippocampus of homozygous mice were significantly higher than those of wild type mice (P < 0.01).The

  20. Vertebrate extracellular preovulatory and postovulatory egg coats.

    Science.gov (United States)

    Menkhorst, Ellen; Selwood, Lynne

    2008-11-01

    Extracellular egg coats deposited by maternal or embryonic tissues surround all vertebrate conceptuses during early development. In oviparous species, the time of hatching from extracellular coats can be considered equivalent to the time of birth in viviparous species. Extracellular coats must be lost during gestation for implantation and placentation to occur in some viviparous species. In the most recent classification of vertebrate extracellular coats, Boyd and Hamilton (Cleavage, early development and implantation of the egg. In: Parkes AS (ed.), Marshall's Physiology of Reproduction, vol. 2, 3rd ed. London: Longmans, Green & Co; 1961:1-126) defined the coat synthesized by the oocyte during oogenesis as primary and the coat deposited by follicle cells surrounding the oocyte as secondary. Tertiary egg coats are those synthesized and deposited around the primary or secondary coat by the maternal reproductive tract. This classification is difficult to reconcile with recent data collected using modern molecular biological techniques that can accurately establish the site of coat precursor synthesis and secretion. We propose that a modification to the classification by Boyd and Hamilton is required. Vertebrate egg coats should be classed as belonging to the following two broad groups: the preovulatory coat, which is deposited during oogenesis by the oocyte or follicle cells, and the postovulatory coats, which are deposited after fertilization by the reproductive tract or conceptus. This review discusses the origin and classification of vertebrate extracellular preovulatory and postovulatory coats and illustrates what is known about coat homology between the vertebrate groups.

  1. Modeling extracellular field potentials and the frequency-filtering properties of extracellular space

    CERN Document Server

    Bedard, C; Destexhe, A; Bédard, Claude; Kroeger, Helmut; Destexhe, Alain

    2003-01-01

    Extracellular local field potentials (LFP) are usually modeled as arising from a set of current sources embedded in a homogeneous extracellular medium. Although this formalism can successfully model several properties of LFPs, it does not account for their frequency-dependent attenuation with distance, a property essential to correctly model extracellular spikes. Here we derive expressions for the extracellular potential that include this frequency-dependent attenuation. We first show that, if the extracellular conductivity is non-homogeneous, there is induction of non-homogeneous charge densities which may result in a low-pass filter. We next derive a simplified model consisting of a punctual (or spherical) current source with spherically-symmetric conductivity/permittivity gradients around the source. We analyze the effect of different radial profiles of conductivity and permittivity on the frequency-filtering behavior of this model. We show that this simple model generally displays low-pass filtering behav...

  2. A continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

    OpenAIRE

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-01-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear...

  3. 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

    OpenAIRE

    Bunney, Tom D.; van Walraven, Hendrika S.; de Boer, Albertus H.

    2001-01-01

    Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our und...

  4. Transport of cisplatin by the copper efflux transporter ATP7B.

    Science.gov (United States)

    Safaei, Roohangiz; Otani, Shinji; Larson, Barrett J; Rasmussen, Michael L; Howell, Stephen B

    2008-02-01

    ATP7B is a P-type ATPase that mediates the efflux of copper. Recent studies have demonstrated that ATP7B regulates the cellular efflux of cisplatin (DDP) and controls sensitivity to the cytotoxic effects of this drug. To determine whether DDP is a substrate for ATP7B, DDP transport was assayed in vesicles isolated from Sf9 cells infected with a baculovirus that expressed either the wild-type ATP7B or a mutant ATP7B that was unable to transport copper as a result of conversion of the transmembrane metal binding CPC motif to CPA. Only the wild-type ATP7B-expressing vesicles exhibited copper-dependent ATPase activity, copper-induced acyl-phosphate formation, and ATP-dependent transport of copper. The amount of DDP that became bound was higher for vesicles expressing either type of ATP7B than for those not expressing either form of ATP7B, but only the vesicles expressing wild-type ATP7B mediated ATP-dependent accumulation of the drug. At pH 4.6, the vesicles expressing the wild-type ATP7B exhibited ATP-dependent accumulation of DDP with an apparent K(m) of 1.2 +/- 0.5 (S.E.M.) muM and V(max) of 0.03 +/- 0.002 (S.E.M.) nmol/mg of protein/min. DDP also induced the acyl-phosphorylation of ATP7B but at a much slower rate than copper. Copper and DDP each inhibited the ATP-dependent transport of the other. These results establish that DDP is a substrate for ATP7B but is transported at a much slower rate than copper.

  5. Extracellular proteolysis in the adult murine brain.

    Science.gov (United States)

    Sappino, A P; Madani, R; Huarte, J; Belin, D; Kiss, J Z; Wohlwend, A; Vassalli, J D

    1993-08-01

    Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

  6. The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2011-01-01

    conditions in order to verify intrinsic impairments. To resemble dynamic equilibrium present in whole cells between ATP synthesis and utilization, ATP was measured in the presence of an ATP consuming enzyme, hexokinase, under steady state. Mitochondria were isolated using an affinity based method which...... or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n=20 in each group), precultured under normophysiological...... selects the mitochondria based on an antibody recognizing the mitochondrial outer membrane and not by size through gradient centrifugation. The dynamic equilibrium between ATP synthesis and ATP consumption is 35% lower in isolated mitochondria from myotubes established from type 2 diabetic subjects...

  7. Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5'-triphosphate (ATP).

    Science.gov (United States)

    Sharma, Piyush S; Dabrowski, Marcin; Noworyta, Krzysztof; Huynh, Tan-Phat; Kc, Chandra B; Sobczak, Janusz W; Pieta, Piotr; D'Souza, Francis; Kutner, Wlodzimierz

    2014-09-24

    For molecular imprinting of oxidatively electroactive analytes by electropolymerization, we used herein reductively electroactive functional monomers. As a proof of concept, we applied C60 fullerene adducts as such for the first time. For that, we derivatized C60 to bear either an uracil or an amide, or a carboxy addend for recognition of the adenosine-5'-triphosphate (ATP) oxidizable analyte with the ATP-templated molecularly imprinted polymer (MIP-ATP). Accordingly, the ATP complex with all of the functional monomers formed in solution was potentiodynamically electropolymerized to deposit an MIP-ATP film either on an Au electrode of the quartz crystal resonator or on a Pt disk electrode for the piezoelectric microgravimetry (PM) or capacitive impedimetry (CI) determination of ATP, respectively, under the flow-injection analysis (FIA) conditions. The apparent imprinting factor for ATP was ∼4.0. After extraction of the ATP template, analytical performance of the resulting chemosensors, including detectability, sensitivity, and selectivity, was characterized. The limit of detection was 0.3 and 0.03mM ATP for the PM and CI chemosensor, respectively. The MIP-ATP film discriminated structural analogues of ATP quite well. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the experimental data of the ATP sorption and sorption stability constants appeared to be nearly independent of the adopted sorption model.

  8. 15 CFR 295.11 - Technical and educational services for ATP recipients.

    Science.gov (United States)

    2010-01-01

    ... ATP recipients. 295.11 Section 295.11 Commerce and Foreign Trade Regulations Relating to Commerce and... PROGRAMS ADVANCED TECHNOLOGY PROGRAM General § 295.11 Technical and educational services for ATP recipients... National Institute of Standards and Technology. (c) From time to time, ATP may conduct public workshops...

  9. 7 CFR 3300.88 - Fees for U.S. ATP certificates.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Fees for U.S. ATP certificates. 3300.88 Section 3300... EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP); INSPECTION, TESTING, AND CERTIFICATION OF SPECIAL EQUIPMENT Other Provisions § 3300.88 Fees for U.S. ATP certificates. The fee schedule for issuance of U.S....

  10. Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

    DEFF Research Database (Denmark)

    Liu, Jianming; Kandasamy, Vijayalakshmi; Würtz, Anders

    2016-01-01

    Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F1-ATPase...

  11. Glycolysis and ATP degradation in cod ( Gadus morhua ) at subzero temperatures in relation to thaw rigor

    DEFF Research Database (Denmark)

    Cappeln, Gertrud; Jessen, Flemming

    2001-01-01

    Glycolysis was shown to occur during freezing of cod of decrease in glycogen and an increase in lactate. In addition, the ATP content decreased during freezing. Synthesis of ATP was measured as degradation of glycogen. During storage at -9 and - 12 degreesC it was found that degradation of ATP...

  12. Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

    NARCIS (Netherlands)

    Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C. D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

    2014-01-01

    Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we

  13. Strain background modifies phenotypes in the ATP8B1-deficient mouse

    NARCIS (Netherlands)

    Shah, S.; Sanford, U.R.; Vargas, J.C.; Xu, H.; Groen, A.; Paulusma, C.C.; Grenert, J.P.; Pawlikowska, L.; Sen, S.; Oude Elferink, R.P.J.; Bull, L.N.

    2010-01-01

    BACKGROUND: Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficie

  14. Bioluminescence microscopy: application to ATP measurements in single living cells

    Science.gov (United States)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  15. ATP binding to a multisubunit enzyme: statistical thermodynamics analysis

    CERN Document Server

    Zhang, Yunxin

    2012-01-01

    Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical thermodynamics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provided a new way to understand biophysical processes by statistical thermodynamics analysis.

  16. Students' Interdisciplinary Reasoning about "High-Energy Bonds" and ATP

    CERN Document Server

    Dreyfus, Benjamin W; Sawtelle, Vashti; Svoboda, Julia; Turpen, Chandra; Redish, Edward F

    2012-01-01

    Students' sometimes contradictory ideas about ATP (adenosine triphosphate) and the nature of chemical bonds have been studied in the biology and chemistry education literatures, but these topics are rarely part of the introductory physics curriculum. We present qualitative data from an introductory physics course for undergraduate biology majors that seeks to build greater interdisciplinary coherence and therefore includes these topics. In these data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see that students' perceptions of how each scientific discipline bounds the system of interest can influence how they justify their reasoning about a topic that crosses disciplines. This has consequences for a vision of interdisciplinary education that respects disciplinary perspectives while bringing them into interaction in ways that demonstrate consistency amongst the perspectiv...

  17. Structure and mechanism of ATP-dependent phospholipid transporters

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Poulsen, Lisbeth Rosager; Bailly, Aurélien

    2015-01-01

    Background ATP-binding cassette (ABC) transporters and P4-ATPases are two large and seemingly unrelated families of primary active pumps involved in moving phospholipids from one leaflet of a biological membrane to the other. Scope of review This review aims to identify common mechanistic features...... in the way phospholipid flipping is carried out by two evolutionarily unrelated families of transporters. Major conclusions Both protein families hydrolyze ATP, although they employ different mechanisms to use it, and have a comparable size with twelve transmembrane segments in the functional unit. Further......, despite differences in overall architecture, both appear to operate by an alternating access mechanism and during transport they might allow access of phospholipids to the internal part of the transmembrane domain. The latter feature is obvious for ABC transporters, but phospholipids and other hydrophobic...

  18. ATP6V1G1 — EDRN Public Portal

    Science.gov (United States)

    ATP6V1G1 is a subunit of vacuolar ATPase (V-ATPase), a multisubunit enzyme. V-ATPase is an enzyme transporter that functions to acidify intracellular compartments in eukaryotic cells. This acidification process is necessary for such intracellular processes as protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation. V-ATPase is ubiquitously expressed and is present in endomembrane organelles such as vacuoles, lysosomes, endosomes, the Golgi apparatus, chromaffin granules and coated vesicles, as well as in the plasma membrane. V-ATPase is composed of a cytosolic V1 domain and a transmembrane V0 domain. The V1 domain consists of three A, three B, and two G subunits, as well as a C, D, E, F, and H subunit. The V1 domain contains the ATP catalytic site.

  19. Responses of Rat P2X2 Receptors to Ultrashort Pulses of ATP Provide Insights into ATP Binding and Channel Gating

    Science.gov (United States)

    Moffatt, Luciano; Hume, Richard I.

    2007-01-01

    To gain insight into the way that P2X2 receptors localized at synapses might function, we explored the properties of outside-out patches containing many of these channels as ATP was very rapidly applied and removed. Using a new method to calibrate the speed of exchange of solution over intact patches, we were able to reliably produce applications of ATP lasting <200 μs. For all concentrations of ATP, there was a delay of at least 80 μs between the time when ATP arrived at the receptor and the first detectable flow of inward current. In response to 200-μs pulses of ATP, the time constant of the rising phase of the current was ∼600 μs. Thus, most channel openings occurred when no free ATP was present. The current deactivated with a time constant of ∼60 ms. The amplitude of the peak response to a brief pulse of a saturating concentration of ATP was ∼70% of that obtained during a long application of the same concentration of ATP. Thus, ATP leaves fully liganded channels without producing an opening at least 30% of the time. Extensive kinetic modeling revealed three different schemes that fit the data well, a sequential model and two allosteric models. To account for the delay in opening at saturating ATP, it was necessary to incorporate an intermediate closed state into all three schemes. These kinetic properties indicate that responses to ATP at synapses that use homomeric P2X2 receptors would be expected to greatly outlast the duration of the synaptic ATP transient produced by a single presynaptic spike. Like NMDA receptors, P2X2 receptors provide the potential for complex patterns of synaptic integration over a time scale of hundreds of milliseconds. PMID:17664346

  20. Catalytic strategy used by the myosin motor to hydrolyze ATP.

    Science.gov (United States)

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

  1. Alternative mitochondrial functions in cell physiopathology: beyond ATP production

    Directory of Open Access Journals (Sweden)

    Kowaltowski A.J.

    2000-01-01

    Full Text Available It is well known that mitochondria are the main site for ATP generation within most tissues. However, mitochondria also participate in a surprising number of alternative activities, including intracellular Ca2+ regulation, thermogenesis and the control of apoptosis. In addition, mitochondria are the main cellular generators of reactive oxygen species, and may trigger necrotic cell death under conditions of oxidative stress. This review concentrates on these alternative mitochondrial functions, and their role in cell physiopathology.

  2. Persister formation in Staphylococcus aureus is associated with ATP depletion

    Energy Technology Data Exchange (ETDEWEB)

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  3. Honing in on the ATP Release Channel in Taste Cells.

    Science.gov (United States)

    Medler, Kathryn F

    2015-09-01

    Studies over the last 8 years have identified 3 potential channels that appear to release ATP from Type II cells in response to taste stimuli. These studies have taken different methodological approaches but have all provided data supporting their candidate channel as the ATP release channel. These potential channels include Pannexin 1, Connexins (30 and/or 43), and most recently, the Calhm1 channel. Two papers in this issue of Chemical Senses provide compelling new evidence that Pannexin 1 is not the ATP release channel. Tordoff et al. did a thorough behavioral analysis of the Pannexin1 knock out mouse and found that these animals have the same behavioral responses as wild type mice for 7 different taste stimuli that were tested. Vandenbeuch et al. presented an equally thorough analysis of the gustatory nerve responses in the Pannexin1 knock out mouse and found no differences compared with controls. Thus when the role of Pannexin 1 is analyzed at the systems level, it is not required for normal taste perception. Further studies are needed to determine the role of this hemichannel in taste cells.

  4. ATP, P2X receptors and pain pathways.

    Science.gov (United States)

    Ding, Y; Cesare, P; Drew, L; Nikitaki, D; Wood, J N

    2000-07-01

    A role for ATP in nociception and pain induction was proposed on the basis of human psychophysical experiments shortly after the formulation of the purinergic hypothesis. Following the pharmacological definition of distinct P2X and P2Y purinergic receptor subtypes by Burnstock and his collaborators, molecular cloning studies have identified the gene products that underlie the effects of ATP on peripheral sensory neurons. One particular receptor, P2X(3), is of particular interest in the context of pain pathways, because it is relatively selectively expressed at high levels by nociceptive sensory neurons. Evidence that this receptor may play a role in the excitation of sensory neurons has recently been complemented by studies that suggest an additional presynaptic role in the regulation of glutamate release from primary afferent neurons in the dorsal horn of the spinal cord. In this brief review, we discuss the present state of knowledge of the role of ATP in pain induction through its action on peripheral P2X receptors.

  5. ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs.

    Science.gov (United States)

    Betzenhauser, Matthew J; Wagner, Larry E; Park, Hyung Seo; Yule, David I

    2009-06-12

    ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

  6. Microcystin-LR acute exposure does not alter in vitro and in vivo ATP, ADP and AMP hydrolysis in adult zebrafish (Danio rerio brain membranes

    Directory of Open Access Journals (Sweden)

    Luiza Wilges Kist

    2012-10-01

    Full Text Available Microcystins (MCs are toxins produced by cyanobacteria during the blooms that could accumulate in aquatic animals and be relocated to higher trophic levels. Adenosine triphosphate (ATP acts as an excitatory neurotransmitter and/or a neuromodulator in the extracellular space playing important roles in physiological and pathological conditions. The aim of this study was, therefore, to evaluate the acute effects of different concentrations of MC-LR on nucleoside triphosphate diphosphohydrolases and 5’-nucleotidade in adult zebrafish (Danio rerio brain membranes. The results have shown no significant changes in ATP, adenosine diphosphate (ADP and adenosine monophosphate (AMP hydrolysis in zebrafish brain membranes. MC-LR in vitro also did not alter ATP, ADP and AMP hydrolysis in the concentrations tested. These findings show that acute exposure to MC-LR did not modulate ectonucleotidase activity in the conditions tested. However, additional studies including chronic exposure should be performed in order to achieve a better understanding about MC-LR toxicity mechanisms in the central nervous system.

  7. Extracellular matrix and tissue engineering applications

    NARCIS (Netherlands)

    Fernandes, Hugo; Moroni, Lorenzo; Blitterswijk, van Clemens; Boer, de Jan

    2009-01-01

    The extracellular matrix is a key component during regeneration and maintenance of tissues and organs, and it therefore plays a critical role in successful tissue engineering as well. Tissue engineers should recognise that engineering technology can be deduced from natural repair processes. Due to a

  8. Extracellular vesicles: fundamentals and clinical relevance

    Directory of Open Access Journals (Sweden)

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  9. Extracellular calmodulin: A polypeptide signal in plants?

    Institute of Scientific and Technical Information of China (English)

    孙大业; 唐文强; 马力耕

    2001-01-01

    Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca2+ signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation,and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore,we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein,phospholipase C, IP3, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.

  10. Fragmentation of extracellular matrix by hypochlorous acid

    DEFF Research Database (Denmark)

    Woods, Alan A; Davies, Michael Jonathan

    2003-01-01

    The interaction of extracellular matrix with cells regulates their adhesion, migration and proliferation, and it is believed that damage to vascular matrix components is a factor in the development of atherosclerosis. Evidence has been provided for a role for the haem enzyme MPO (myeloperoxidase)...

  11. Protons, the thylakoid membrane, and the chloroplast ATP synthase.

    Science.gov (United States)

    Junge, W

    1989-01-01

    According to the chemiosmotic theory, proton pumps and ATP synthases are coupled by lateral proton flow through aqueous phases. Three long-standing challenges to this concept, all of which have been loosely subsumed under 'localized coupling' in the literature, were examined in the light of experiments carried out with thylakoids: (1) Nearest neighbor interaction between pumps and ATP synthases. Considering the large distances between photosystem II and CFoCF1, in stacked thylakoids this is a priori absent. (2) Enhanced proton diffusion along the surface of the membrane. This could not be substantiated for the outer side of the thylakoid membrane. Even for the interface between pure lipid and water, two laboratories have reported the absence of enhanced diffusion. (3) Localized proton ducts in the membrane. Intramembrane domains that can transiently trap protons do exist in thylakoid membranes, but because of their limited storage capacity for protons, they probably do not matter for photophosphorylation under continuous light. Seemingly in favor of localized proton ducts is the failure of a supposedly permeant buffer to enhance the onset lag of photophosphorylation. However, it was found that failure of some buffers and the ability of others in this respect were correlated with their failure/ability to quench pH transients in the thylakoid lumen, as predicted by the chemiosmotic theory. It was shown that the chemiosmotic concept is a fair approximation, even for narrow aqueous phases, as in stacked thylakoids. These are approximately isopotential, and protons are taken in by the ATP synthase straight from the lumen. The molecular mechanism by which F0F1 ATPases couple proton flow to ATP synthesis is still unknown. The threefold structural symmetry of the headpiece that, probably, finds a corollary in the channel portion of these enzymes appeals to the common wisdom that structural symmetry causes functional symmetry. "Rotation catalysis" has been proposed. It is

  12. The Role of Reactive-Oxygen-Species in Microbial Persistence and Inflammation

    Directory of Open Access Journals (Sweden)

    Ralee Spooner

    2011-01-01

    Full Text Available The mechanisms of chronic infections caused by opportunistic pathogens are of keen interest to both researchers and health professionals globally. Typically, chronic infectious disease can be characterized by an elevation in immune response, a process that can often lead to further destruction. Reactive-Oxygen-Species (ROS have been strongly implicated in the aforementioned detrimental response by host that results in self-damage. Unlike excessive ROS production resulting in robust cellular death typically induced by acute infection or inflammation, lower levels of ROS produced by host cells are increasingly recognized to play a critical physiological role for regulating a variety of homeostatic cellular functions including growth, apoptosis, immune response, and microbial colonization. Sources of cellular ROS stimulation can include “danger-signal-molecules” such as extracellular ATP (eATP released by stressed, infected, or dying cells. Particularly, eATP-P2X7 receptor mediated ROS production has been lately found to be a key modulator for controlling chronic infection and inflammation. There is growing evidence that persistent microbes can alter host cell ROS production and modulate eATP-induced ROS for maintaining long-term carriage. Though these processes have yet to be fully understood, exploring potential positive traits of these “injurious” molecules could illuminate how opportunistic pathogens maintain persistence through physiological regulation of ROS signaling.

  13. Extracellular calmodulin: A polypeptide signal in plants?

    Institute of Scientific and Technical Information of China (English)

    SUN; Daye(

    2001-01-01

    [1]Cheng. W. Y., Cyclic 3', 5'-nucleotide phosphodiestrase: demonstration of an activator, Biochm. Biophys. Res. Commun.,1970, 38: 533-538.[2]Boynton, A. L., Whitfield, J. F., MacManus, J. P., Calmodulin stimulates DNA synthesis by rat liver cells, BBRC.1980,95(2): 745-749.[3]Gorbacherskaya, L. V., Borovkova, T. V., Rybin, U. O. et al., Effect of exogenous calmodulin on lymphocyte proliferation in normal subjects, Bull Exp. Med. Biol., 1983, 95: 361-363.[4]Wong, P. Y.-K., Lee, W. H., Chao, PH.-W., The role of calmodulin in prostaglandin metabolism, Ann. NY Acad. Sci.,1980, 356: 179-189.[5]Mac Neil, S., Dawson, R. A., Crocker, G. et al., Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth, J. Invest. Dermatol., 1984, 83: 15-19.[6]Crocker, D. G., Dawson, R. A., Mac Neil, S. et al., An extracellular role for calmodulin-like activity in cell proliferation,Biochem. J., 1988, 253: 877-884.[7]Polito. V. S., Calmodulin and calmodulin inhibitors: effect on pollen germination and tube growth, in Pollen: Biology and Implications for Plant Breeding (eds. Mulvshy, D. L., Ottaviaro, E.), New York: Elsevier, 1983.53-60.[8]Biro, R. L., Sun, D. Y., Roux, S. J.et al., Characterization of oat calmodulin and radioimmunoassay of its subcellular distribution, Plant Physiol., 1984,75: 382-386.[9]Terry, M. E., Bonner, B. A., An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of IAA-induced growth, Plant Physiol., 1980, 66: 321-325.[10]Josefina, H. N., Aldasars, J. J., Rodriguez, D., Localization of calmodulin on embryonic Cice aricium L, in Molecular and Cellular Aspects of Calcium in Plant Development (ed. Trewavas, A. J.), New York, London: Plenum Press, 1985, 313.[11]Dauwalder, M., Roux, S. J., Hardison, L., Distribution of calmodulin in pea seedling: immunocytochemical localization in plumules and root apices, Planta, 1986, 168: 461

  14. Hemolysis is a primary ATP-release mechanism in human erythrocytes

    Science.gov (United States)

    Sikora, Jacek; Orlov, Sergei N.; Furuya, Kishio

    2014-01-01

    The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented. PMID:25097178

  15. Towards a multiscale description of microvascular flow regulation: O2-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks

    Directory of Open Access Journals (Sweden)

    Daniel eGoldman

    2012-07-01

    Full Text Available Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100ms that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2. The model further predicts how insulin, at concentrations found in prediabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by

  16. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    Science.gov (United States)

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  17. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction

    DEFF Research Database (Denmark)

    Kringelbach, Tina M.; Aslan, Derya; Novak, Ivana

    2015-01-01

    effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP...... and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low...... concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling...

  18. Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocities.

    Science.gov (United States)

    Yan, Chen; Shen, Shaochuan; Yun, Junxian; Wang, Lianghua; Yao, Kejian; Yao, Shan-Jing

    2008-12-01

    This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion-exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP-containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high-purity ATP directly from fermentation feedstock at high flow velocities.

  19. Oxidative and other posttranslational modifications in extracellular vesicle biology.

    Science.gov (United States)

    Szabó-Taylor, Katalin; Ryan, Brent; Osteikoetxea, Xabier; Szabó, Tamás G; Sódar, Barbara; Holub, Marcsilla; Németh, Andrea; Pálóczi, Krisztina; Pállinger, Éva; Winyard, Paul; Buzás, Edit I

    2015-04-01

    Extracellular vesicles including exosomes, microvesicles and apoptotic vesicles, are phospholipid bilayer surrounded structures secreted by cells universally, in an evolutionarily conserved fashion. Posttranslational modifications such as oxidation, citrullination, phosphorylation and glycosylation play diverse roles in extracellular vesicle biology. Posttranslational modifications orchestrate the biogenesis of extracellular vesicles. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.

  20. [Stabilization of Cadmium Contaminated Soils by Ferric Ion Modified Attapulgite (Fe/ATP)--Characterizations and Stabilization Mechanism].

    Science.gov (United States)

    Rong, Yang; Li, Rong-bo; Zhou, Yong-li; Chen, Jing; Wang, Lin-ling; Lu, Xiao-hua

    2015-08-01

    Ferric ion modified attapulgite (Fe/ATP) was prepared by impregnation and its structure and morphology were characterized. The toxicity characteristic leaching procedure (TCLP) was used to evaluate the effect of Cadmium( Cd) stabilization in soil with the addition of attapulgite (ATP) and Fe/ATP. The stabilization mechanism of Cd was further elucidated by comparing the morphologies and structure of ATP and Fe/ATP before and after Cd adsorption. Fe/ATP exhibited much better adsorption capacity than ATP, suggesting different adsorption mechanisms occurred between ATP and Fe/ATP. The leaching concentrations of Cd in soil decreased by 45% and 91% respectively, with the addition of wt. 20% ATP and Fe/ATP. The former was attributed to the interaction between Cd2 and --OH groups by chemical binding to form inner-sphere complexes in ATP and the attachment between Cd2+ and the defect sites in ATP framework. Whereas Cd stabilization with Fe/ATP was resulted from the fact that the active centers (--OH bonds or O- sites) on ATP could react with Fe3+ giving Fe--O--Cd-- bridges, which helped stabilize Cd in surface soil. What'more, the ferric oxides and metal hydroxides on the surface of ATP could interact with Cd, probably by the formation of cadmium ferrite. In conclusion, Fe/ATP, which can be easily prepared, holds promise as a potential low-cost and environmental friendly stabilizing agent for remediation of soil contaminated with heavy metals.

  1. Structural characterization of two metastable ATP-bound states of P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Megan L O'Mara

    Full Text Available ATP Binding Cassette (ABC transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs, the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg(2+ with each NBD indicates that the coordination of ATP and Mg(2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation

  2. Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water.

    Science.gov (United States)

    Vang, Óluva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jørgen

    2014-11-01

    Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 10(4) in drinking water, was detected with the ATP assay, as well as 10(2) to 10(3) times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay.

  3. Developmental changes in the expression of ATP7A during a critical period in postnatal neurodevelopment.

    Science.gov (United States)

    Niciu, M J; Ma, X-M; El Meskini, R; Ronnett, G V; Mains, R E; Eipper, B A

    2006-01-01

    ATP7A is a P-type ATPase that transports copper from cytosol into the secretory pathway for loading onto cuproproteins or efflux. Mutations in Atp7a cause Menkes disease, a copper-deficiency disorder fatal in the postnatal period due to severe neurodegeneration. Early postnatal copper injections are known to diminish degenerative changes in some human patients and mice bearing mutations in Atp7a. In situ hybridization studies previously demonstrated that ATP7A transcripts are expressed widely in the brain. ATP7A-specific antibody was used to study the neurodevelopmental expression and localization of ATP7A protein in the mouse brain. Based on immunoblot analyses, ATP7A expression is most abundant in the early postnatal period, reaching peak levels at P4 in neocortex and cerebellum. In the developing and adult brain, ATP7A levels are greatest in the choroid plexus/ependymal cells of the lateral and third ventricles. ATP7A expression decreases in most neuronal subpopulations from birth to adulthood. In contrast, ATP7A expression increases in CA2 hippocampal pyramidal and cerebellar Purkinje neurons. ATP7A is expressed in a subset of astrocytes, microglia, oligodendrocytes, tanycytes and endothelial cells. ATP7A is largely localized to the trans-Golgi network, adopting the cell-specific and developmentally-regulated morphology of this organelle. The presence of ATP7A in the axons of postnatal, but not adult, optic nerve suggests stage-specific roles for this enzyme. In sum, the precisely-regulated neurodevelopmental expression of ATP7A correlates well with the limited therapeutic window for effective treatment of Menkes disease.

  4. Phosphocreatine, an Intracellular High-Energy Compound, is Found in the Extracellular Fluid of the Seminal Vesicles in Mice and Rats

    Science.gov (United States)

    Lee, H. J.; Fillers, W. S.; Iyengar, M. R.

    1988-10-01

    High levels of phosphocreatine, a compound known to serve as an intracellular energy reserve, were found in the fluid contained in seminal vesicle glands. The concentrations of phosphocreatine in the extracellular fluid in the mouse and rat were found to be 5.6 ± 1.6 and 2.2 ± 0.8 μ mol/g, respectively, which are higher than the intracellular levels reported for smooth muscles. The creatine concentrations in the seminal vesicular fluid from these two species were 22.8 ± 3.1 and 13.0 ± 5.3 μ mol/g, respectively. These creatine levels are approximately 100 and 65 times higher than the creatine levels in mammalian blood. Smaller amounts of ATP (phosphocreatine/ATP ratio of 20-40) and traces of ADP were also found. Comparison of the pattern of distribution of macromolecules (proteins and DNA) with the distribution of phosphocreatine between the cells and the fluid of the seminal vesicle indicates that cell lysis did not account for the phosphocreatine in the seminal vesicle fluid. Rather, the available evidence strongly suggests that this high-energy compound is actively secreted. We found that in the testes, the sperm are exposed to the highest known creatine concentration in any mammalian tissue studied. Based on these results and other recent reports, we propose that the extracellular phosphocreatine, ATP, and creatine are involved in sperm metabolism.

  5. Distinct Wilson's disease mutations in ATP7B are associated with enhanced binding to COMMD1 and reduced stability of ATP7B

    NARCIS (Netherlands)

    de Bie, Prim; van de Sluis, Bart; Burstein, Ezra; van den Berghe, Peter V. E.; Muller, Patricia; Berger, Ruud; Gitlin, Jonathan D.; Wijmenga, Cisca; Klomp, Leo W. J.

    2007-01-01

    Background & Aims: Wilson's disease (WD) is characterized by hepatic copper overload and caused by mutations in the gene encoding the copper-transporting P-type adenosine triphospharase (ATPase) ATP7B. ATP7B interacts with COMMD1, a protein that is deleted in Bedlington terriers with hereditary copp

  6. Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2011-01-01

    The copper-transporting P1B-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mut

  7. Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

    DEFF Research Database (Denmark)

    Beuschlein, Felix; Boulkroun, Sheerazed; Osswald, Andrea

    2013-01-01

    Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na+/K+ ATPase α...... subunit) and ATP2B3 (encoding a Ca2+ ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced...... affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation...

  8. Involvement of extracellular matrix constituents in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  9. Terrestrial evolution of polymerization of amino acids - Heat to ATP

    Science.gov (United States)

    Fox, S. W.; Nakashima, T.

    1981-01-01

    Sets of amino acids containing sufficient trifunctional monomer are thermally polymerized at temperatures such as 65 deg; the amino acids order themselves. Various polymers have diverse catalytic activities. The polymers aggregate, in aqueous solution, to cell-like structures having those activities plus emergent properties, e.g. proliferatability. Polyamino acids containing sufficient lysine catalyze conversion of free amino acids, by ATP, to small peptides and a high molecular weight fraction. The lysine-rich proteinoid is active in solution, within suspensions of cell-like particles, or in other particles composed of lysine-rich proteinoid and homopolyribonucleotide. Selectivities are observed. An archaic polyamino acid prelude to coded protein synthesis is indicated.

  10. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    Science.gov (United States)

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  11. ATP11B Mediates Platinum Resistance in Ovarian Cancer

    Science.gov (United States)

    2013-05-01

    rather than cations) from the outer and inner leaf- let of membrane bilayers (13, 17–23). Although the putative role of ATP11B, based on its protein... membrane protein 4 is implicated in trans-Golgi network vesicle traf- ficking. Mol Biol Cell. 1999;10(6):1957–1972. 29. Ohmichi M, Hayakawa J, Tasaka K...isolation and character- ization of cholesterol-rich membrane domains from trans-Golgi network vesicles . J Lipid Res. 2011; 52(3):582–589.

  12. Extracellular matrix component signaling in cancer

    DEFF Research Database (Denmark)

    Multhaupt, Hinke A. B.; Leitinger, Birgit; Gullberg, Donald

    2016-01-01

    Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motil...... as well as matrix constitution and protein crosslinking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression. [on SciFinder(R)]......Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization...... and motility but also provides survival and proliferation cues. The major classes of cell surface receptors for matrix macromols. are the integrins, discoidin domain receptors, and transmembrane proteoglycans such as syndecans and CD44. Cells respond not only to specific ligands, such as collagen, fibronectin...

  13. Biotechnological Aspects of Microbial Extracellular Electron Transfer

    Science.gov (United States)

    Kato, Souichiro

    2015-01-01

    Extracellular electron transfer (EET) is a type of microbial respiration that enables electron transfer between microbial cells and extracellular solid materials, including naturally-occurring metal compounds and artificial electrodes. Microorganisms harboring EET abilities have received considerable attention for their various biotechnological applications, in addition to their contribution to global energy and material cycles. In this review, current knowledge on microbial EET and its application to diverse biotechnologies, including the bioremediation of toxic metals, recovery of useful metals, biocorrosion, and microbial electrochemical systems (microbial fuel cells and microbial electrosynthesis), were introduced. Two potential biotechnologies based on microbial EET, namely the electrochemical control of microbial metabolism and electrochemical stimulation of microbial symbiotic reactions (electric syntrophy), were also discussed. PMID:26004795

  14. Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

    Institute of Scientific and Technical Information of China (English)

    ZENG Xiaomei; SHI Xiaobing; SHEN Yungang

    2004-01-01

    The ε subunit of the chloroplast ATP synthase and the truncated ε mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coli. When the ε subunit or the truncated ε proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of ε-deficient membrane reconstituted with the ε subunit or the truncated ε proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ε protein. These results suggested that: (a) the N-terminal domain of the ε subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ε subunit; and (b) the C-terminal domain of the ε subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.

  15. Tetraspanins in Extracellular Vesicle Formation and Function

    OpenAIRE

    Andreu, Zoraida; Yáñez-Mó, María

    2014-01-01

    Extracellular vesicles (EVs) represent a novel mechanism of intercellular communication as vehicles for intercellular transfer of functional membrane and cytosolic proteins, lipids, and RNAs. Microvesicles, ectosomes, shedding vesicles, microparticles, and exosomes are the most common terms to refer to the different kinds of EVs based on their origin, composition, size, and density. Exosomes have an endosomal origin and are released by many different cell types, participating in different phy...

  16. Tetraspanins in Extracellular Vesicle formation and function

    OpenAIRE

    Zoraida Andreu Martínez; María eYáñez-Mó

    2014-01-01

    Extracellular vesicles (EVs) represent a novel mechanism of intercellular communication as vehicles for intercellular transfer of functional membrane and cytosolic proteins, lipids, and RNAs. Microvesicles, ectosomes, shedding vesicles, microparticles and exosomes are the most common terms to refer to the different kinds of EVs based on their origin, composition, size and density. Exosomes have an endosomal origin and are released by many different cell types, participating in different physi...

  17. Matrix Extracellular Phosphoglycoprotein Inhibits Phosphate Transport

    OpenAIRE

    Marks, J; Churchill, L J; Debnam, E. S.; Unwin, R J

    2008-01-01

    The role of putative humoral factors, known as phosphatonins, in phosphate homeostasis and the relationship between phosphate handling by the kidney and gastrointestinal tract are incompletely understood. Matrix extracellular phosphoglycoprotein (MEPE), one of several candidate phosphatonins, promotes phosphaturia, but whether it also affects intestinal phosphate absorption is unknown. Here, using the in situ intestinal loop technique, we demonstrated that short-term infusion of MEPE inhibits...

  18. Engineering hydrogels as extracellular matrix mimics

    OpenAIRE

    Geckil, Hikmet; Xu, Feng; Zhang, Xiaohui; Moon, SangJun; Demirci, Utkan

    2010-01-01

    Extracellular matrix (ECM) is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth and cell–cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable....

  19. How reliable are ATP bioluminescence meters in assessing decontamination of environmental surfaces in healthcare settings?

    Directory of Open Access Journals (Sweden)

    Navid Omidbakhsh

    Full Text Available BACKGROUND: Meters based on adenosine triphosphate (ATP bioluminescence measurements in relative light units (RLU are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry? OBJECTIVE: This study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals' interference in their readings. METHODS: First, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter's ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings. RESULTS: All four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated. CONCLUSIONS: Apart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries.

  20. Insights into the subunit in-teractions of the chloroplast ATP synthase

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  1. Induction of Posttranslational Modifications of Mitochondrial Proteins by ATP Contributes to Negative Regulation of Mitochondrial Function.

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    Full Text Available It is generally accepted that ATP regulates mitochondrial function through the AMPK signaling pathway. However, the AMPK-independent pathway remains largely unknown. In this study, we investigated ATP surplus in the negative regulation of mitochondrial function with a focus on pyruvate dehydrogenase (PDH phosphorylation and protein acetylation. PDH phosphorylation was induced by a high fat diet in the liver of obese mice, which was associated with ATP elevation. In 1c1c7 hepatoma cells, the phosphorylation was induced by palmitate treatment through induction of ATP production. The phosphorylation was associated with a reduction in mitochondria oxygen consumption after 4 h treatment. The palmitate effect was blocked by etomoxir, which inhibited ATP production through suppression of fatty acid β-oxidation. The PDH phosphorylation was induced by incubation of mitochondrial lysate with ATP in vitro without altering the expression of PDH kinase 2 (PDK2 and 4 (PDK4. In addition, acetylation of multiple mitochondrial proteins was induced by ATP in the same conditions. Acetyl-CoA exhibited a similar activity to ATP in induction of the phosphorylation and acetylation. These data suggest that ATP elevation may inhibit mitochondrial function through induction of the phosphorylation and acetylation of mitochondrial proteins. The results suggest an AMPK-independent mechanism for ATP regulation of mitochondrial function.

  2. Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

    Science.gov (United States)

    Rice, Austin J; Harrison, Alistair; Alvarez, Frances J D; Davidson, Amy L; Pinkett, Heather W

    2014-05-23

    Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.

  3. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    NARCIS (Netherlands)

    Tjaden, J.; Haferkamp, I.; Boxma, B.; Tielens, A.G.; Huynen, M.A.; Hackstein, J.H.P.

    2004-01-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-ge

  4. Changes of IK,ATP current density and allosteric modulation during chronic atrial fibrillation

    Institute of Scientific and Technical Information of China (English)

    WU Gang; HUANG Cong-xin; TANG Yan-hong; JIANG Hong; WAN Jun; CHEN Hui; XIE Qiang; HUANG Zheng-rong

    2005-01-01

    Background Atrial fibrillation (AF) is the most common supraventricular arrhythmia in clinical practice. Chronic atrial fibrillation (CAF) is associated with ionic remodeling. However, little is known about the activity of ATP-sensitive potassium current (IK,ATP) during CAF. So we studied the changes of IK,ATP density and allosteric modulation of ATP-sensitivity by intracellular pH during CAF.Methods Myocardium samples were obtained from the right auricular appendage of patients with rheumatic heart disease complicated with valvular disease in sinus rhythm (SR) or CAF. There were 14 patients in SR group and 9 patients in CAF group. Single atrial cells were isolated using an enzyme dispersion technique. IK,ATP was recorded using the whole-cell and inside-out configuration of voltage-clamp techniques. In whole-cell model, myocytes of SR and CAF groups were perfused with simulated ischemic solution to elicit IK,ATP. In inside-out configuration, the internal patch membranes were exposed to different ATP concentrations in pH 7.4 and 6.8.Results Under simulated ischemia, IK,ATP current density of CAF group was significantly higher than in SR group [(83.5±10.8) vs. (58.7±8.4) pA/pF, P<0.01]. IK,ATP of the two groups showed ATP concentration-dependent inhibition. The ATP concentration for 50% current inhibition (IC50) for the SR group was significantly different in pH 7.4 and pH 6.8 (24 vs. 74 μmol/L, P<0.01). The IC50 did not change significantly in CAF group when the pH decreased from 7.4 to 6.8.Conclusions During CAF, IK,ATP current density was increased and its allosteric modulation of ATP-sensitivity by intracellular pH was diminished.

  5. Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms

    Science.gov (United States)

    Gorgojo, Juan; Scharrig, Emilia; Gómez, Ricardo M.; Harvill, Eric T.; Rodríguez, Maria Eugenia

    2017-01-01

    B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts. PMID:28095485

  6. Extracellular quality control in the epididymis

    Institute of Scientific and Technical Information of China (English)

    Gail A. Cornwall; H. Henning von Horsten; Douglas Swartz; Seethal Johnson; Kim Chau; Sandra Whelly

    2007-01-01

    The epididymal lumen represents a unique extracellular environment because of the active sperm maturation process that takes place within its confines. Although much focus has been placed on the interaction of epididymal secretory proteins with spermatozoa in the lumen, very little is known regarding how the complex epididymal milieu as a whole is maintained, including mechanisms to prevent or control proteins that may not stay in their native folded state following secretion. Because some misfolded proteins can form cytotoxic aggregate structures known as amyloid, it is likely that control/surveillance mechanisms exist within the epididymis to protect against this process and allow sperm maturation to occur. To study protein aggregation and to identify extracellular quality control mechanisms in the epididymis, we used the cystatin family of cysteine protease inhibitors, including cystatin-related epididymal spermatogenic and cystatin C as molecular models because both proteins have inherent properties to aggregate and form amyloid. In this chapter, we present a brief summary of protein aggregation by the amyloid pathway based on what is known from other organ systems and describe quality control mechanisms that exist intracellularly to control protein misfolding and aggregation. We then present a summary of our studies of cystatinrelated epididymal spermatogenic (CRES) oligomerization within the epididymal lumen, including studies suggesting that transglutaminase cross-linking may be one mechanism of extracellular quality control within the epididymis.

  7. EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

    Directory of Open Access Journals (Sweden)

    A. V. Oberemko

    2014-12-01

    Full Text Available This review presents a generalized definition of vesicles as bilayer extracellular organelles of all celular forms of life: not only eu-, but also prokaryotic. The structure and composition of extracellular vesicles, history of research, nomenclature, their impact on life processes in health and disease are discussed. Moreover, vesicles may be useful as clinical instruments for biomarkers, and they are promising as biotechnological drug. However, many questions in this area are still unresolved and need to be addressed in the future. The most interesting from the point of view of practical health care represents a direction to study the effect of exosomes and microvesicles in the development and progression of a particular disease, the possibility of adjusting the pathological process by means of extracellular vesicles of a particular type, acting as an active ingredient. Relevant is the further elucidation of the role and importance of exosomes to the surrounding cells, tissues and organs at the molecular level, the prospects for the use of non-cellular vesicles as biomarkers of disease.

  8. Extracellular conversion of adiponectin hexamers into trimers

    Science.gov (United States)

    Kim, Jeong-a; Nuñez, Martha; Briggs, David B.; Laskowski, Bethany L.; Chhun, Jimmy J.; Eleid, Joseph K.; Quon, Michael J.; Tsao, Tsu-Shuen

    2012-01-01

    Adiponectin is an adipocyte-secreted hormone that exists as trimers, hexamers and larger species collectively referred to as HMW (high-molecular-weight) adiponectin. Whether hexamers or HMW adiponectin serve as precursors for trimers outside the circulation is currently unknown. Here, we demonstrate that adiponectin trimers can be generated from larger oligomers secreted from primary rat adipose cells or differentiated 3T3-L1 adipocytes. Purified hexameric, but not HMW, adiponectin converted into trimers in conditioned media separated from 3T3-L1 adipocytes or, more efficiently, when enclosed in the dialysis membrane in the presence of adipocytes. Several lines of evidence indicate that the conversion is mediated by an extracellular redox system. First, N-terminal epitope-tagged hexamers converted into trimers without proteolytic removal of the tag. Secondly, appearance of trimers was associated with conversion of disulfide-bonded dimers into monomers. Thirdly, thiol-reactive agents inhibited conversion into trimers. Consistent with a redox-based mechanism, purified hexamers reductively converted into trimers in defined glutathione redox buffer with reduction potential typically found in the extracellular environment while the HMW adiponectin remained stable. In addition, conversion of hexamers into trimers was enhanced by NADPH, but not by NADP+. Collectively, these data strongly suggest the presence of an extracellular redox system capable of converting adiponectin oligomers. PMID:22973892

  9. Extracellular DNA: the tip of root defenses?

    Science.gov (United States)

    Hawes, Martha C; Curlango-Rivera, Gilberto; Wen, Fushi; White, Gerard J; Vanetten, Hans D; Xiong, Zhongguo

    2011-06-01

    This review discusses how extracellular DNA (exDNA) might function in plant defense, and at what level(s) of innate immunity this process might operate. A new role for extracellular factors in mammalian defense has been described in a series of studies. These studies reveal that cells including neutrophils, eosinophils, and mast cells produce 'extracellular traps' (ETs) consisting of histone-linked exDNA. When pathogens are attracted to such ETs, they are trapped and killed. When the exDNA component of ETs is degraded, trapping is impaired and resistance against invasion is reduced. Conversely, mutation of microbial genes encoding exDNases that degrade exDNA results in loss of virulence. This discovery that exDNases are virulence factors opens new avenues for disease control. In plants, exDNA is required for defense of the root tip. Innate immunity-related proteins are among a group of >100 proteins secreted from the root cap and root border cell populations. Direct tests revealed that exDNA also is rapidly synthesized and exported from the root tip. When this exDNA is degraded by the endonuclease DNase 1, root tip resistance to fungal infection is lost; when the polymeric structure is degraded more slowly, by the exonuclease BAL31, loss of resistance to fungal infection is delayed accordingly. The results suggest that root border cells may function in a manner analogous to that which occurs in mammalian cells.

  10. Extracellular matrix proteins involved in pseudoislets formation.

    Science.gov (United States)

    Maillard, Elisa; Sencier, Marie-Christine; Langlois, A; Bietiger, William; Krafft, Mp; Pinget, Michel; Sigrist, Séverine

    2009-01-01

    Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.

  11. Intracellular Biopotentials During Static Extracellular Stimulation

    Science.gov (United States)

    Klee, Maurice

    1973-01-01

    Two properties of the intracellular potentials and electric fields resulting from static extracellular stimulation are obtained for arbitrarily shaped cells. First, the values of intracellular potential are shown to be bounded by the maximum and minimum values of extracellular potential on the surface of the cell. Second, the volume average of the magnitude of intracellular electric field is shown to have an upper bound given by the ratio of the magnitude of the largest extracellular potential difference on the surface of the cell to a generalized length constant λ = [σintraVcell/(σmemb Acell)]1/2, where Vcell and Acell are the volume and surface area of the cell, σintra is the intracellular conductivity (reciprocal ohms per centimeter), and σmemb is the membrane conductivity (reciprocal ohms per square centimeter). The use of the upper bound on the volume average of the magnitude of intracellular electric field as an estimate for intracellular isopotentiality is discussed and the use of the generalized length constant for electrically describing arbitrary cells is illustrated for cylindrical- and spheroidal-shaped cells. PMID:4726882

  12. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

    Directory of Open Access Journals (Sweden)

    Joseph E Ippolito

    Full Text Available Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5 on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.

  13. Genetic variation in ATP5O is associated with skeletal muscle ATP50 mRNA expression and glucose uptake in young twins.

    Directory of Open Access Journals (Sweden)

    Tina Rönn

    Full Text Available BACKGROUND: Impaired oxidative capacity of skeletal muscle mitochondria contribute to insulin resistance and type 2 diabetes (T2D. Furthermore, mRNA expression of genes involved in oxidative phosphorylation, including ATP5O, is reduced in skeletal muscle from T2D patients. Our aims were to investigate mechanisms regulating ATP5O expression in skeletal muscle and association with glucose metabolism, and the relationship between ATP5O single nucleotide polymorphisms (SNPs and risk of T2D. METHODOLOGY/PRINCIPAL FINDINGS: ATP5O mRNA expression was analyzed in skeletal muscle from young (n = 86 and elderly (n = 68 non-diabetic twins before and after a hyperinsulinemic euglycemic clamp. 11 SNPs from the ATP5O locus were genotyped in the twins and a T2D case-control cohort (n = 1466. DNA methylation of the ATP5O promoter was analyzed in twins (n = 22 using bisulfite sequencing. The mRNA level of ATP5O in skeletal muscle was reduced in elderly compared with young twins, both during basal and insulin-stimulated conditions (p<0.0005. The degree of DNA methylation around the transcription start of ATP5O was <1% in both young and elderly twins and not associated with mRNA expression (p = 0.32. The mRNA level of ATP5O in skeletal muscle was positively related to insulin-stimulated glucose uptake (regression coefficient = 6.6; p = 0.02. Furthermore, two SNPs were associated with both ATP5O mRNA expression (rs12482697: T/T versus T/G; p = 0.02 and rs11088262: A/A versus A/G; p = 0.004 and glucose uptake (rs11088262: A/A versus A/G; p = 0.002 and rs12482697: T/T versus T/G; p = 0.005 in the young twins. However, we could not detect any genetic association with T2D. CONCLUSIONS/SIGNIFICANCE: Genetic variation and age are associated with skeletal muscle ATP5O mRNA expression and glucose disposal rate, suggesting that combinations of genetic and non-genetic factors may cause the reduced expression of ATP5O in T2D muscle. These findings propose a role for ATP5O, in

  14. ATP citrate lyase inhibitors as novel cancer therapeutic agents.

    Science.gov (United States)

    Zu, Xu-Yu; Zhang, Qing-Hai; Liu, Jiang-Hua; Cao, Ren-Xian; Zhong, Jing; Yi, Guang-Hui; Quan, Zhi-Hua; Pizzorno, Giuseppe

    2012-05-01

    ATP citrate lyase (ACL or ACLY) is an extra-mitochondrial enzyme widely distributed in various human and animal tissues. ACL links glucose and lipid metabolism by catalyzing the formation of acetyl-CoA and oxaloacetate from citrate produced by glycolysis in the presence of ATP and CoA. ACL is aberrantly expressed in many immortalized cells and tumors, such as breast, liver, colon, lung and prostate cancers, and is correlated reversely with tumor stage and differentiation, serving as a negative prognostic marker. ACL is an upstream enzyme of the long chain fatty acid synthesis, providing acetyl-CoA as an essential component of the fatty acid synthesis. Therefore, ACL is a key enzyme of cellular lipogenesis and potent target for cancer therapy. As a hypolipidemic strategy of metabolic syndrome and cancer treatment, many small chemicals targeting ACL have been designed and developed. This review article provides an update for the research and development of ACL inhibitors with a focus on their patent status, offering a new insight into their potential application.

  15. Application of ATP measurements to the microbiological evaluation of a petroleum reservoir

    Energy Technology Data Exchange (ETDEWEB)

    Jones, P.M.

    1981-06-01

    The objective of the work reported in this document was to determine whether the bioluminescent luciferin/luciferase based adenosine triphosphate (ATP) assay could be used as a rapid field tests for determining the presence and numbers of microorganisms in oil field fluids. The ATP-photometric technique employed is based on the ATP-mediated bioluminescent oxidation of firefly luciferin. Light production is stoichiometrically related to ATP concentration; ATP concentration is related to numbers of living organisms present in a sample. Samples used in this study comprised reservoir fluids collected from several Southern California oilfields. Based on experimental evidence, it was concluded that the ATP assay could be profitably applied to Microbially Enhanced Oil Recovery (MEOR) process monitoring and control. The theoretical basis for the assay, field-usage methodologies, and fundamentals of data interpretation are presented to make the document usable as a field manual.

  16. ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    obese and eight subjects with type 2 diabetes precultured under normophysiological conditions. Furthermore, mitochondria were isolated and ATP production was measured by luminescence at baseline and during acute insulin stimulation with or without concomitant ATP utilization by hexokinase. Mitochondrial...... mass and the ATP synthesis rate, neither at baseline nor during acute insulin stimulation, were not different between groups. The ratio of ATP synthesis rate at hexokinase versus ATP synthesis rate at baseline was lower in diabetic mitochondria compared to lean mitochondria. Thus the lower content...... of muscle mitochondria in type 2 diabetes in vivo is an adaptive trait and mitochondrial dysfunction in type 2 diabetes in vivo is based both on primarily impaired ATP synthesis and an adaptive loss of mitochondrial mass....

  17. Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease.

    LENUS (Irish Health Repository)

    Pitceathly, Robert D S

    2012-09-11

    Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN).

  18. ATP consumption of eukaryotic flagella measured at a single-cell level

    CERN Document Server

    Chen, Daniel T N; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-01-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. Here, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axonemes ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ~2.3e5 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating wavef...

  19. Glutamate and ATP at the Interface Between Signaling and Metabolism in Astroglia

    DEFF Research Database (Denmark)

    Parpura, Vladimir; Fisher, Elizabeth S; Lechleiter, James D

    2017-01-01

    Glutamate is the main excitatory transmitter in the brain, while ATP represents the most important energy currency in any living cell. Yet, these chemicals play an important role in both processes, enabling them with dual-acting functions in metabolic and intercellular signaling pathways. Glutamate...... can fuel ATP production, while ATP can act as a transmitter in intercellular signaling. We discuss the interface between glutamate and ATP in signaling and metabolism of astrocytes. Not only do glutamate and ATP cross each other's paths in physiology of the brain, but they also do so in its pathology....... We present the fabric of this process in (patho)physiology through the discussion of synthesis and metabolism of ATP and glutamate in astrocytes as well as by providing a general description of astroglial receptors for these molecules along with the downstream signaling pathways that may be activated...

  20. Crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus.

    Science.gov (United States)

    Manjula, M; Pampa, K J; Kumar, S M; Mukherjee, S; Kunishima, N; Rangappa, K S; Lokanath, N K

    2015-03-27

    The ATP binding cassette (ABC) transporters, represent one of the largest superfamilies of primary transporters, which are very essential for various biological functions. The crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus has been determined at 1.77 Å resolution. The crystal structure revealed that the protomer has two thick arms, (arm I and II), which resemble 'L' shape. The ATP-binding pocket is located close to the end of arm I. ATP molecule is docked into the active site of the protein. The dimeric crystal structure of ATP-binding subunit of ABC transporter from G. kaustophilus has been compared with the previously reported crystal structure of ATP-binding subunit of ABC transporter from Salmonella typhimurium.

  1. Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

    Science.gov (United States)

    Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

    2012-01-01

    Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

  2. Structure-guided simulations illuminate the mechanism of ATP transport through VDAC1.

    Science.gov (United States)

    Choudhary, Om P; Paz, Aviv; Adelman, Joshua L; Colletier, Jacques-Philippe; Abramson, Jeff; Grabe, Michael

    2014-07-01

    The voltage-dependent anion channel (VDAC) mediates the flow of metabolites and ions across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, whereas the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics simulations to construct a Markov state model of ATP permeation. These simulations indicate that ATP flows through VDAC through multiple pathways, in agreement with our structural data and experimentally determined physiological rates.

  3. The interference of HEPES buffer during amperometric detection of ATP in clinical applications.

    Science.gov (United States)

    Masson, Jean-Francois; Gauda, Estelle; Mizaikoff, Boris; Kranz, Christine

    2008-04-01

    HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, (31)P NMR, and (1)H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca(2+) in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.

  4. Genetic effects of ATP1A2 in familial hemiplegic migraine type II and animal models.

    Science.gov (United States)

    Gritz, Stephanie M; Radcliffe, Richard A

    2013-04-05

    Na(+)/K(+)-ATPase alpha 2 (Atp1a2) is an integral plasma membrane protein belonging to the P-type ATPase family that is responsible for maintaining the sodium (Na(+)) and potassium (K(+)) gradients across cellular membranes with hydrolysis of ATP. Atp1a2 contains two subunits, alpha and beta, with each having various isoforms and differential tissue distribution. In humans, mutations in ATP1A2 are associated with a rare form of hereditary migraines with aura known as familial hemiplegic migraine type II. Genetic studies in mice have revealed other neurological effects of Atp1a2 in mice including anxiety, fear, and learning and motor function disorders. This paper reviews the recent findings in the literature concerning Atp1a2.

  5. A futile cycle, formed between two ATP-dependant -glutamyl cycle enzymes, -glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

    Indian Academy of Sciences (India)

    Akhilesh Kumar; Anand Kumar Bachhawat

    2010-03-01

    Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the -glutamyl cycle leads to ATP depletion. The enzyme -glutamyl cysteine synthetase (-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, -glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

  6. Periodate oxidized ATP (oATP) reduces hyperalgesia in mice: involvement of P2X7 receptors and implications for therapy.

    Science.gov (United States)

    Fulgenzi, A; Ticozzi, P; Gabel, C A; Dell'Antonio, G; Quattrini, A; Franzone, J S; Ferrero, M E

    2008-01-01

    Some inflammatory mediators play an important role not only in the pathogenesis of the inflammatory pain, but also in that of neuropathic and visceral pain. We previously showed the antihyperalgesic effect of oATP, the inhibitor of the P2X7 receptors for the pro-nociceptive ATP, in experimental inflammation. Here we show the antihyperalgesic effect of oATP in mouse models of neuropathic and visceral pain, other than in a model of arthritic pain mimicking rheumatoid arthritis in humans. We also show that mice lacking P2X7 receptors (KO) are resistant to hyperalgesic thermal stimuli following the induction of arthritic, neuropathic and visceral pain. Local (injection into the right hind paw) pre-treatment with oATP is able to prevent the successive induction of ATP-dependent hyperalgesia in wild type mice. In addition, KO mice are not insensitive to intraplantar treatment with ATP. Our data suggest that, even if oATP is able to inhibit purinoceptors different from P2X7, the latter are the more important involved in pain transmission.

  7. ATP sensitizes H460 lung carcinoma cells to cisplatin-induced apoptosis.

    Science.gov (United States)

    Swennen, Els L R; Ummels, Vanessa; Buss, Irina; Jaehde, Ulrich; Bast, Aalt; Dagnelie, Pieter C

    2010-03-30

    Platinum resistance of cancer cells may evolve due to a decrease in intracellular drug accumulation, decreased cell permeability or by an increased deactivation of the drug by glutathione (GSH). The aim of this study was (1) to investigate the effect of adenosine 5'-triphosphate (ATP) on the cytotoxicity of cisplatin in a large cell lung carcinoma cell line (H460), and (2) to examine the potential involvement of increased cisplatin uptake, GSH depletion and pyrimidine starvation by ATP in this effect. H460 cells were harvested and seeded (5% CO(2); 37 degrees C). Subsequently, cells were incubated with medium or ATP followed by an incubation with cisplatin. Cytotoxicity screening was analyzed by the sulforhodamine B (SRB) colorimetric assay, lactate dehydrogenase and caspase-3/7 activity. Pre-incubation for 72h with 0.3 and 3mM ATP strongly enhanced the anti-proliferative potency of cisplatin 2.9- and 7.6-fold, respectively. Moreover, after incubation of H460 cells with 0.3mM ATP the intracellular platinum concentration increased, indicating increased cisplatin uptake by ATP. ATP, despite lowering the LD(50) of cisplatin, did not modulate GSH levels in H460 cells. ATP itself showed a biphasic effect on H460 cell growth: 0.3mM inhibited H460 cell growth via the pyrimidine starvation effect, activation of caspase-3/7 and LDH leakage, while 3mM ATP showed no effect on cell growth. In conclusion, ATP sensitizes the H460 cells to cisplatin-induced apoptosis. The effect of 0.3mM ATP is not due to GSH depletion but involves increased cisplatin uptake and pyrimidine starvation due to ATP conversion to adenosine followed by cellular uptake.

  8. ATP7A (Menkes Protein) functions in Axonal Targeting and Synaptogenesis

    OpenAIRE

    Meskini, Rajaâ El; Crabtree, Kelli L.; Cline, Laura B.; Mains, Richard E.; Eipper, Betty A.; Ronnett, Gabriele V.

    2007-01-01

    Menkes Disease (MD) is a neurodegenerative disorder caused by mutations in the copper transporter, ATP7A, a P-type ATPase. We previously used the olfactory system to demonstrate that ATP7A expression is developmentally, not constitutive, regulated, peaking during synaptogenesis when it is highly expressed in extending axons in a copper-independent manner. Although not known to be associated with axonal functions, we explored the possibility that the inability of mutant ATP7A to support axon o...

  9. Is ATP a central synaptic mediator for certain primary afferent fibers from mammalian skin?

    OpenAIRE

    Fyffe, R E; Perl, E R

    1984-01-01

    The possibility that ATP acts as a synaptic mediator at the central terminals of primary afferent fibers was examined by applying it iontophoretically to neurons of the outer layers of the cat spinal cord in vivo. ATP proved to be selectively excitatory for a limited subset of spinal neurons. Those units consistently excited by ATP iontophoresis with very small currents (2-15 nA) responded to gentle mechanical stimulation of the skin and usually evidenced excitatory input from unmyelinated pr...

  10. Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+ signaling in bovine vascular endothelial cells.

    Science.gov (United States)

    Aromolaran, Ademuyiwa S; Zima, Aleksey V; Blatter, Lothar A

    2007-07-01

    The role of glycolytically generated ATP in Ca(2+)/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca(2+) signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca(2+) concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + beta-hydroxybutyrate (beta-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca(2+)](i). The rise of [Ca(2+)](i) was characterized by a transient spike followed by a small sustained plateau of elevated [Ca(2+)](i). In the absence of extracellular Ca(2+) 2-DG caused an increase in [Ca(2+)](i), suggesting that inhibition of glycolysis directly triggered release of Ca(2+) from intracellular endoplasmic reticulum (ER) Ca(2+) stores. The inositol-1,4,5-trisphosphate receptor (IP(3)R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca(2+) response. Ca(2+) release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca(2+)](i) wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca(2+)](i). Propagating [Ca(2+)](i) waves were preceded by [Ca(2+)](i) oscillations and small, highly localized elevations of [Ca(2+)](i) (Ca(2+) puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca(2+) response, and vice versa during inhibition of CaMKII 2-DG-induced Ca(2+) release was attenuated. Similar results were obtained with pyr + beta-HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg(2+) concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca(2+) release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP(3)R.

  11. Inhibition of ATP Hydrolysis by Thermoalkaliphilic F1Fo-ATP Synthase Is Controlled by the C Terminus of the ɛ Subunit

    OpenAIRE

    2006-01-01

    The F1Fo-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F1 moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F1 complexes from this thermoalkaliphile. Like the native F1Fo-ATP synthase, the recombinant TA2F1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent ...

  12. The a subunit asymmetry dictates the two opposite rotation directions in the synthesis and hydrolysis of ATP by the mitochondrial ATP synthase.

    Science.gov (United States)

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2015-01-01

    The main and best known role of the mitochondrial ATP synthase is to synthesize ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. However, under different conditions, the same enzyme can also switch to the opposite function of ATP hydrolysis and exploits its energy to pump protons against their gradient and energize the membrane. The change in functionality is linked to the change of direction of rotation of the two matched sectors of this unique complex, namely the hydrophilic F1, which performs the catalysis, and the hydrophobic membrane-embedded FO, which channels protons. Accordingly, viewed from the matrix side, ATP synthesis is driven by counterclockwise rotation and ATP hydrolysis by clockwise rotation of the FO rotor which is transmitted to F1. ATP dissipation through this mechanism features some diseases such as myocardial ischemia. Increasing evidence shoulders the hypothesis that the asymmetry of the a subunit of FO and particularly the steric arrangement of the two inner semi-channels for protons, play a key role in conferring to the coupled bi-functional complex the ability to reverse rotation by switching from ATP synthesis to ATP hydrolysis and vice versa. Accordingly, the conserved steric arrangement of the chiral a subunit of FO yields the same direction of rotation for all the ATP synthases. According to this hypothesis, the a subunit chirality imposes the direction of rotation of the rotor according to the proton gradient across the membrane. It seems likely that the direction of rotation of the membrane-embedded c-ring, which is adjacent to the a-subunit and acts as a rotor, may be under multiple control, being rotation essential to make the whole enzyme machinery work. However, the asymmetric features of the a subunit would make it the master regulator, thus directly determining which of the two functions, ATP production or ATP dissipation, will be performed. The handedness of a subunit should

  13. The Role of ATP in Mechanically Stimulated Rapid Closure of the Venus's Flytrap.

    Science.gov (United States)

    Jaffe, M J

    1973-01-01

    When the midribs of untreated traps of Dionaea muscipula are frozen in liquid nitrogen after rapid closure, they contain significantly less ATP than those frozen before closure. Exogenous ATP causes a significant increase in the rate of mechanically stimulated trap closure. Illuminated traps close faster than those kept in the dark. The traps of plants placed in 100% O(2) close much faster than do air controls, while 100% CO(2) inhibits closure. It is concluded that ATP is probably the native source of potential energy for contraction of the trap's midrib, and that if the endogenous ATP titer is increased by oxidative phosphorylation or an exogenous source, the trap will close faster.

  14. Rapid antimicrobial susceptibility determination of uropathogens in clinical urine specimens by use of ATP bioluminescence.

    Science.gov (United States)

    Ivancic, Vesna; Mastali, Mitra; Percy, Neil; Gornbein, Jeffrey; Babbitt, Jane T; Li, Yang; Landaw, Elliot M; Bruckner, David A; Churchill, Bernard M; Haake, David A

    2008-04-01

    We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

  15. Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase.

    Science.gov (United States)

    Cong, Weitao; Zhao, Ting; Zhu, Zhongxin; Huang, Binbin; Ma, Weide; Wang, Yuehui; Tan, Yi; Chakrabarti, Subrata; Li, Xiaokun; Jin, Litai; Cai, Lu

    2014-04-01

    Mitochondrial ATP production is the main energy source for the cell. Diabetes reduces the efficient generation of ATP, possibly due to the inactivation of ATP synthase. However, the exact mechanism by which diabetes induces inactivation of ATP synthase remains unknown, as well as whether such inactivation has a role in the development of pathological abnormalities of the diabetic heart. To address these issues, we used cardiac metallothionein-transgenic (MT-TG) and wild-type (WT) mice with streptozotocin-induced diabetes, since we have demonstrated previously that diabetes-induced cardiac damage and remodeling were found in WT diabetic mice, but not in MT-TG diabetic mice. Immunohistochemical and biochemical assays were used to compare pathological and biochemical changes of the heart between MT-TG and WT diabetic mice, and a proteomic assay to evaluate ATP synthase expression and tyrosine nitration, with its activity. LC/MS analysis revealed that diabetes increased tyrosine nitration of the ATP synthase α subunit at Tyr(271), Tyr(311), and Tyr(476), and the β subunit at Tyr(269) and Tyr(508), and also significantly reduced ATP synthase activity by ~32%. These changes were not observed in MT-TG diabetic mice. Furthermore, parallel experiments with induced expression of cardiac MT by zinc supplementation in diabetic mice produced similar effects. These results suggest that MT can preserve ATP synthase activity in streptozotocin-induced diabetes, probably through the inhibition of ATP synthase nitration. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. atp mutants of Escherichia coli fail to grow on succinate due to a transport deficiency

    DEFF Research Database (Denmark)

    Boogerd, Fred; Boe, Lars; Michelsen, Ole

    1998-01-01

    Escherichia coli atp mutants, which lack a functional Hf-ATPase complex, are capable of growth on glucose but not on succinate or other C-4-dicarboxylates (Suc(-) phenotype). Suc(+) revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack......) was expressed in trans, the Suc(-) phenotype of the atp deletion strain reverted to Suc(+), which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C-4-dicarboxylates is insufficient transport capacity for these substrates....

  17. atp mutants of Escherichia coli fail to grow on succinate due to a transport deficiency

    DEFF Research Database (Denmark)

    Boogerd, Fred; Boe, Lars; Michelsen, Ole

    1998-01-01

    Escherichia coli atp mutants, which lack a functional Hf-ATPase complex, are capable of growth on glucose but not on succinate or other C-4-dicarboxylates (Suc(-) phenotype). Suc(+) revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack......) was expressed in trans, the Suc(-) phenotype of the atp deletion strain reverted to Suc(+), which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C-4-dicarboxylates is insufficient transport capacity for these substrates....

  18. De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

    DEFF Research Database (Denmark)

    Heinzen, Erin L; Swoboda, Kathryn J; Hitomi, Yuki

    2012-01-01

    in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without...... affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3....

  19. Pharmacological and molecular comparison of K(ATP) channels in rat basilar and middle cerebral arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Edvinsson, Lars; Olesen, Jes;

    2006-01-01

    ATP-sensitive potassium (K(ATP)) channels play an important role in the regulation of cerebral vascular tone. In vitro studies using synthetic K(ATP) channel openers suggest that the pharmacological profiles differ between rat basilar arteries and rat middle cerebral arteries. To address this issue....... Densitometric measurements of the Western blot signals further showed higher expression levels of Kir6.1 and SUR2B proteins in rat middle cerebral arteries than was found in rat basilar arteries. In conclusion, our in vitro pharmacological studies showed no evidence for functional endothelial K(ATP) channels...

  20. ESR-spektroskopische Untersuchungen der F0F1-ATP-Synthase aus Escherichia coli

    OpenAIRE

    Motz, Christian

    1999-01-01

    Die FoF1-ATP-Synthase katalysiert die Synthese von ATP aus ADP und Pi bei der oxidativen bzw. Photophosphorylierung. Der ATP-Synthase-Komplex läßt sich in zwei funktionelle Einheiten unterteilen: Fo ist ein integraler Membranproteinkomplex, der den Protonenkanal bildet. F1 hingegen ist ein wasserlöslicher Proteinkomplex, der die Nukleotidbindungsstellen trägt. Die ATP-Synthase aus Escherichia coli hat die Zusammensetzung alpha3beta3gamma delta epsilon für die F1 und ab2c9-12 für den Fo-Teil. ...

  1. Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases.

    Directory of Open Access Journals (Sweden)

    Andrea Seper

    Full Text Available The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

  2. Extracellular polymeric substances play roles in extracellular electron transfer of Shewanella oneidensis MR-1

    DEFF Research Database (Denmark)

    Xiao, Yong; Zhang, En-Hua; Christensen, Hans Erik Mølager

    It is well known that microorganism is surrounded by extracellular polymeric substances (EPS) which include polysaccharides, proteins, glycoproteins, nucleic acids, phospholipids, and humic acids. However, previous studies on microbial extracellular electron transfer (EET) are conducted on cells...... the extraction (Figure 1.a and 1.b). Comparing to cells in control group, MR-1 treated at 38 °C for EPS extraction showed different electrochemical characterizations as revealed by differential pulse voltammetry (Figure 1.c). EPS extracted from MR-1 also was proved to be electrochemically active. The present...

  3. Resorbable extracellular matrix grafts in urologic reconstruction

    Directory of Open Access Journals (Sweden)

    Richard A. Santucci

    2005-06-01

    Full Text Available PURPOSE: There is an increasingly large body of literature concerning tissue-engineering products that may be used in urology. Some of these are quite complex (such as multilayer patient-specific cell-seeded implants yet the most simple and successful products to date are also the most uncomplicated: resorbable acellular extra-cellular matrices (ECMs harvested from animals. ECMs have been used in a variety of difficult urologic reconstruction problems, and this review is intended to summarize this complex literature for the practicing urologist. METHODS: Medline search of related terms such as "SIS, small intestinal submucosa, ECM, extracellular matrix, acellular matrix and urologic reconstruction". Manuscripts missed in the initial search were taken from the bibliographies of the primary references. RESULTS: Full review of potential clinical uses of resorbable extra-cellular matrices in urologic reconstruction. CONCLUSIONS: Currently, the "state of the art" in tissue engineering solutions for urologic reconstruction means resorbable acellular xenograft matrices. They show promise when used as a pubovaginal sling or extra bolstering layers in ureteral or urethral repairs, although recent problems with inflammation following 8-ply pubovaginal sling use and failures after 1- and 4-ply SIS repair of Peyronie's disease underscore the need for research before wide adoption. Preliminary data is mixed concerning the potential for ECM urethral patch graft, and more data is needed before extended uses such as bladder augmentation and ureteral replacement are contemplated. The distant future of ECMs in urology likely will include cell-seeded grafts with the eventual hope of producing "off the shelf" replacement materials. Until that day arrives, ECMs only fulfill some of the requirements for the reconstructive urologist.

  4. Managing Brain Extracellular K(+) during Neuronal Activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

    2016-01-01

    reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia...... parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na(+)/K(+)-ATPase in the regulation of extracellular K(+) in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better...

  5. Biogenesis, delivery, and function of extracellular RNA

    Directory of Open Access Journals (Sweden)

    James G. Patton

    2015-08-01

    Full Text Available The Extracellular RNA (exRNA Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA.

  6. Extracellular polymeric bacterial coverages as minimal surfaces

    CERN Document Server

    Saa, A; Saa, Alberto; Teschke, Omar

    2005-01-01

    Surfaces formed by extracellular polymeric substances enclosing individual and some small communities of {\\it Acidithiobacillus ferrooxidans} on plates of hydrophobic silicon and hydrophilic mica are analyzed by means of atomic force microscopy imaging. Accurate nanoscale descriptions of such coverage surfaces are obtained. The good agreement with the predictions of a rather simple but realistic theoretical model allows us to conclude that they correspond, indeed, to minimal area surfaces enclosing a given volume associated with the encased bacteria. This is, to the best of our knowledge, the first shape characterization of the coverage formed by these biomolecules, with possible applications to the study of biofilms.

  7. Bidirectional extracellular matrix signaling during tissue morphogenesis

    Science.gov (United States)

    Gjorevski, Nikolce; Nelson, Celeste M.

    2009-01-01

    Normal tissue development and function are regulated by the interplay between cells and their surrounding extracellular matrix (ECM). The ECM provides biochemical and mechanical contextual information that is conveyed from the cell membrane through the cytoskeleton to the nucleus to direct cell phenotype. Cells, in turn, remodel the ECM and thereby sculpt their local microenvironment. Here we review the mechanisms by which cells interact with, respond to, and influence the ECM, with particular emphasis placed on the role of this bidirectional communication during tissue morphogenesis. We also discuss the implications for successful engineering of functional tissues ex vivo. PMID:19896886

  8. NASA ATP Force Measurement Technology Capability Strategic Plan

    Science.gov (United States)

    Rhew, Ray D.

    2008-01-01

    The Aeronautics Test Program (ATP) within the National Aeronautics and Space Administration (NASA) Aeronautics Research Mission Directorate (ARMD) initiated a strategic planning effort to re-vitalize the force measurement capability within NASA. The team responsible for developing the plan included members from three NASA Centers (Langley, Ames and Glenn) as well as members from the Air Force s Arnold Engineering and Development Center (AEDC). After visiting and discussing force measurement needs and current capabilities at each participating facility as well as selected force measurement companies, a strategic plan was developed to guide future NASA investments. This paper will provide the details of the strategic plan and include asset management, organization and technology research and development investment priorities as well as efforts to date.

  9. ATP-dependent potassium channels and type 2 diabetes mellitus.

    Science.gov (United States)

    Bonfanti, Dianne Heloisa; Alcazar, Larissa Pontes; Arakaki, Priscila Akemi; Martins, Laysa Toschi; Agustini, Bruna Carla; de Moraes Rego, Fabiane Gomes; Frigeri, Henrique Ravanhol

    2015-05-01

    Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus.

  10. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...... to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations...

  11. Analysis on Phase Transformation (ATP) Using Computational Thermal Principles (CTP)

    Institute of Scientific and Technical Information of China (English)

    N.Alagurmurthi; K.Palaniradja; V. Soundararajan

    2004-01-01

    Computer analysis based on computational thermal principles to predict the transformation kinetics in steels at varying temperatures is of great practical importance in different areas of heat treatment. As a result, using the theory of transient state heat conduction with convective boundary conditions, an efficient program named "ATP" (Analysis on Phase Transformation) has been developed to determine the temperature distribution under different quenching conditions for different geometries such as plate, cylinder and sphere. In addition to these the microstructures and the corresponding hardness developed during quenching are predicted using Time Temperature Transformation (TTT) diagram incorporated in the analysis. To approve our work, dilation curves, Heisler charts and time-temperature history curve have been generated. This paper deals with basic objective of the program (ATP) determination of temperature, microstructure and hardness distribution and also includes an online prediction of austenite-pearlite and austenite-martensite transformation in steels along with the corresponding retained fractions. The quenching of a cylinder in gases, liquids and liquid metals is analyzed to show the non-liner effect of cylinder diameter on the temperature and microstructures. Further in the program we have considered a typical 1080 steel cylinders quenched in water for predicting and comparing the program results with experimental values and can be extended even to other grades of steels. The numerical results of program are found to be in good agreement with the experimental data obtained. Finally the quenching process analysis described in the study appears to be a promising tool for the design of heat-treatment process parameters for steels.

  12. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2012-01-01

    , but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production...... satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential...... in the single mitochondrion in response to decreased mitochondrial mass and reduced extra-mitochondrial energy supply. This then can lead to the increased damage of DNA, lipids and proteins of the mitochondria as postulated by the free radical theory of aging....

  13. Deletion of a unique loop in the mycobacterial F‐ATP synthase γ subunit sheds light on its inhibitory role in ATP hydrolysis‐driven H+ pumping

    National Research Council Canada - National Science Library

    Hotra, Adam; Suter, Manuel; Biuković, Goran; Ragunathan, Priya; Kundu, Subhashri; Dick, Thomas; Grüber, Gerhard

    2016-01-01

    The F 1 F O ‐ ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F...

  14. Mesurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Ytting, Cecilie Karkov; Fuglsang, Anja Thoe; Hiltunen, J. Kalervo

    2012-01-01

    Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F0F1 ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating...... of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal...... activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose...

  15. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  16. Urinary ATP may be a dynamic biomarker of detrusor overactivity in women with overactive bladder syndrome.

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    Miguel Silva-Ramos

    Full Text Available BACKGROUND: Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function and in the pathophysiology of detrusor overactivity. ATP mediates nonadrenergic-noncholinergic detrusor contractions in overactive bladders. In vitro studies have demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladder samples (OAB release more ATP than controls. Here, we compared the urinary ATP concentration in samples collected non-invasively from OAB women with detrusor overactivity and age-matched controls. METHODS: Patients with neurologic diseases, history of malignancy, urinary tract infections or renal impairment (creatinine clearance <70 ml/min were excluded. All patients completed a 3-day voiding diary, a 24 h urine collection and blood sampling to evaluate creatinine clearance. Urine samples collected during voluntary voids were immediately freeze-preserved for ATP determination by the luciferin-luciferase bioluminescence assay; for comparison purposes, samples were also tested for urinary nerve growth factor (NGF by ELISA. RESULTS: The urinary content of ATP, but not of NGF, normalized to patients' urine creatinine levels (ATP/Cr or urinary volume (ATP.Vol were significantly (P<0.05 higher in OAB women with detrusor overactivity (n = 34 than in healthy controls (n = 30. Significant differences between the two groups were still observed by boosting urinary ATP/Cr content after water intake, but these were not detected for NGF/Cr. In OAB patients, urinary ATP/Cr levels correlated inversely with mean voided volumes determined in a 3-day voiding diary. CONCLUSION: A high area under the receiver operator characteristics (ROC curve (0.741; 95% CI 0.62-0.86; P<0.001 is consistent with urinary ATP/Cr being a highly sensitive dynamic biomarker for assessing detrusor overactivity in women with OAB syndrome.

  17. Macula densa basolateral ATP release is regulated by luminal [NaCl] and dietary salt intake.

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    Komlosi, Peter; Peti-Peterdi, Janos; Fuson, Amanda L; Fintha, Attila; Rosivall, Laszlo; Bell, Phillip Darwin

    2004-06-01

    One component of the macula densa (MD) tubuloglomerular feedback (TGF) signaling pathway may involve basolateral release of ATP through a maxi-anion channel. Release of ATP has previously been studied during a maximal luminal NaCl concentration ([NaCl](L)) stimulus (20-150 mmol/l). Whether MD ATP release occurs during changes in [NaCl](L) within the physiological range (20-60 mmol/l) has not been examined. Also, because TGF is known to be enhanced by low dietary salt intake, we examined the pattern of MD ATP release from salt-restricted rabbits. Fluorescence microscopy, with fura 2-loaded cultured mouse mesangial cells as biosensors, was used to assess ATP release from the isolated, perfused thick ascending limb containing the MD segment. The mesangial biosensor cells, which contain purinergic receptors and elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) on ATP binding, were placed adjacent to the MD basolateral membrane. Elevations in [NaCl](L) between 0 and 80 mmol/l, in 20-mmol/l increments, caused stepwise increases in [Ca(2+)](i), with the highest increase at [NaCl](L) of approximately 60 mmol/l. Luminal furosemide at 10(-4) mol/l blocked ATP release, which suggests that the efflux of ATP required MD Na-2Cl-K cotransport. A low-salt diet for 1 wk increased the magnitude of [NaCl](L)-dependent elevations in biosensor [Ca(2+)](i) by twofold, whereas high-salt intake had no effect. In summary, ATP release occurs over the same range of [NaCl](L) (20-60 mmol/l) previously reported for TGF responses, and, similar to TGF, ATP release was enhanced by dietary salt restriction. Thus these two findings are consistent with the role of MD ATP release as a signaling component of the TGF pathway.

  18. Global gene profiling of aging lungs in Atp8b1 mutant mice

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    Soundararajan, Ramani; Stearns, Timothy M.; Czachor, Alexander; Fukumoto, Jutaro; Turn, Christina; Westermann-Clark, Emma; Breitzig, Mason; Tan, Lee; Lockey, Richard F.; King, Benjamin L.; Kolliputi, Narasaiah

    2016-01-01

    Objective Recent studies implicate cardiolipin oxidation in several age-related diseases. Atp8b1 encoding Type 4 P-type ATPases is a cardiolipin transporter. Mutation in Atp8b1 gene or inflammation of the lungs impairs the capacity of Atp8b1 to clear cardiolipin from lung fluid. However, the link between Atp8b1 mutation and age-related gene alteration is unknown. Therefore, we investigated how Atp8b1 mutation alters age-related genes. Methods We performed Affymetrix gene profiling of lungs isolated from young (7-9 wks, n=6) and aged (14 months, 14 M, n=6) C57BL/6 and Atp8b1 mutant mice. In addition, Ingenuity Pathway Analysis (IPA) was performed. Differentially expressed genes were validated by quantitative real-time PCR (qRT-PCR). Results Global transcriptome analysis revealed 532 differentially expressed genes in Atp8b1 lungs, 157 differentially expressed genes in C57BL/6 lungs, and 37 overlapping genes. IPA of age-related genes in Atp8b1 lungs showed enrichment of Xenobiotic metabolism and Nrf2-mediated signaling pathways. The increase in Adamts2 and Mmp13 transcripts in aged Atp8b1 lungs was validated by qRT-PCR. Similarly, the decrease in Col1a1 and increase in Cxcr6 transcripts was confirmed in both Atp8b1 mutant and C57BL/6 lungs. Conclusion Based on transcriptome profiling, our study indicates that Atp8b1 mutant mice may be susceptible to age-related lung diseases. PMID:27689529

  19. HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal K ATP channels.

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    Zhang, Hai Xia; Akrouh, Alejandro; Kurata, Harley T; Remedi, Maria Sara; Lawton, Jennifer S; Nichols, Colin G

    2011-03-01

    Murine ventricular and atrial ATP-sensitive potassium (K(ATP)) channels contain different sulfonylurea receptors (ventricular K(ATP) channels are Kir6.2/SUR2A complexes, while atrial K(ATP) channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K(ATP) channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K(ATP) channels over atrial K(ATP) channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as (86)Rb(+) efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K(ATP) currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K(ATP) currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits (86)Rb(+) effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K(ATP) channels.

  20. Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse

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    Vargas, Julie C.; Xu, Hongmei; Groen, Annamiek; Paulusma, Coen C.; Grenert, James P.; Pawlikowska, Ludmila; Sen, Saunak; Elferink, Ronald P. J. Oude; Bull, Laura N.

    2010-01-01

    Background Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. Methodology/Principal Findings We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. Conclusions/Significance Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease. PMID:20126555

  1. Strain background modifies phenotypes in the ATP8B1-deficient mouse.

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    Sohela Shah

    Full Text Available BACKGROUND: Mutations in ATP8B1 (FIC1 underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis to intermittent (benign recurrent intrahepatic cholestasis. The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6, 129, and (B6-129 F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.

  2. Defining the extracellular matrix using proteomics

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    Byron, Adam; Humphries, Jonathan D; Humphries, Martin J

    2013-01-01

    The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems-level understanding of ECM biology. PMID:23419153

  3. Thermoreversible copolymer gels for extracellular matrix.

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    Vernon, B; Kim, S W; Bae, Y H

    2000-07-01

    To improve the properties of a reversible synthetic extracellular matrix based on a thermally reversible polymer, copolymers of N-isopropylacrylamide and acrylic acid were prepared in benzene with varying contents of acrylic acid (0 to 3%) and the thermal properties were evaluated. The poly(N-isopropylacrylamide) and copolymers made with acrylic acid had molecular weights from 0.8 to 1.7 x10(6) D. Differential scanning calorimetry (DSC) showed the high-molecular-weight acrylic acid copolymers had similar onset temperatures to the homopolymers, but the peak width was considerably increased with increasing acrylic acid content. DSC and cloud point measurements showed that polymers with 0 to 3% acrylic acid exhibit a lower critical solution temperature (LCST) transition between 30 degrees and 37 degrees C. In swelling studies, the homopolymer showed significant syneresis at temperatures above 31 degrees C. Copolymers with 1 and 1.5% showed syneresis beginning at 32 degrees and 37 degrees C, respectively. At 37 degrees C the copolymers with 1.5-3% acrylic acid showed little or no syneresis. Due to the high water content and a transition near physiologic conditions (below 37 degrees C), the polymers with 1.5-2.0% acrylic acid exhibited properties that would be useful in the development of a refillable synthetic extracellular matrix. Such a matrix could be applied to several cell types, including islets of Langerhans, for a biohybrid artificial pancreas.

  4. [Glutamic acid as a universal extracellular signal].

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    Yoneda, Yukio

    2015-08-01

    The prevailing view is that both glutamic (Glu) and gamma-aminobutyric (GABA) acids play a role as an amino acid neurotransmitter released from neurons. However, little attention has been paid to the possible expression and functionality of signaling machineries required for amino acidergic neurotransmission in cells other than central neurons. In line with our first demonstration of the presence of Glu receptors outside the brain, in this review I will outline our recent findings accumulated since then on the physiological and pathological significance of neuronal amino acids as an extracellular signal essential for homeostasis in a variety of phenotypic cells. In undifferentiated neural progenitor cells, for instance, functional expression is seen with different signaling machineries used for glutamatergic and GABAergic neurotransmission in neurons. Moreover, Glu plays a role in mechanisms underlying suppression of proliferation for self-replication in undifferentiated mesenchymal stem cells. There is more accumulating evidence for neuronal amino acids playing a role as an extracellular autocrine or paracrine signal commonly used in different phenotypic cells. Evaluation of drugs currently used could be thus beneficial for the efficient prophylaxis and/or the therapy of a variety of diseases relevant to disturbance of amino acid signaling in diverse organs.

  5. Extracellular superoxide dismutase of boar seminal plasma.

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    Kowalowka, M; Wysocki, P; Fraser, L; Strzezek, J

    2008-08-01

    Superoxide dismutase (SOD) is an enzymatic component of the antioxidant defense system that protects spermatozoa by catalysing the dismutation of superoxide anions to hydrogen peroxide and oxygen. Age and season effects on SOD activity in the seminal plasma were measured in boars at the onset of 8 months through a 35-month period. It was found that age-related changes in SOD activity in the seminal plasma were markedly higher in boars less than 2 years of age. However, it appeared that SOD activity was established at the early sexual maturity age (8-12 months). There were variations in SOD activity throughout the season, being significantly higher in spring and autumn than in summer. A secretory extracellular form of SOD (EC-SOD) was purified to homogeneity (350-fold) from boar seminal plasma, using a three-step purification protocol (affinity chromatography followed by ion exchange and ceramic hydroxyapatite chromatography). The molecular properties and specificity of SOD (molecular mass, isoelectric point, optimum pH, thermostability and susceptibility to inhibitors) confirmed that the purified enzyme is an extracellular form of Cu/Zn-superoxide dismutase occurring in boar seminal plasma. The results of this study indicate that EC-SOD is an important antioxidant enzyme of boar seminal plasma, which plays an important physiological role in counteracting oxidative stress in spermatozoa.

  6. Extracellular polymers of ozonized waste activated sludge.

    Science.gov (United States)

    Liu, J C; Lee, C H; Lai, J Y; Wang, K C; Hsu, Y C; Chang, B V

    2001-01-01

    Effect of ozonation on characteristics of waste activated sludge was investigated in the current study. Concentrations of cell-bound extracellular polymers (washed ECPs) did not change much upon ozonation, whereas the sum of cell-bound and soluble extracellular polymers (unwashed ECPs) increased with increasing ozone dose. Washed ECPs in original sludge as divided by molecular weight distribution was 39% 10,000 Da (high MW). It was observed that the low-MW fraction decreased, and the high-MW fraction increased in ozonized sludge. The unwashed ECPs were characterized as 44% in low MW, 30% in medium MW, and 26% in high MW. Both low-MW and medium-MW fractions of unwashed ECPs decreased while high-MW fraction increased in ozonized sludge. The dewaterability of ozonized sludge, assessed by capillary suction time (CST) and specific resistance to filtration (SRF), deteriorated with ozone dose. The optimal dose of cationic polyelectrolyte increased with increasing ozone dose. The production rate and the accumulated amount of methane gas of ozonized sludge were also higher.

  7. Molecular dynamics simulation studies of GLUT4: substrate-free and substrate-induced dynamics and ATP-mediated glucose transport inhibition.

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    Suma Mohan

    Full Text Available BACKGROUND: Glucose transporter 4 (GLUT4 is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states. Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. CONCLUSIONS/SIGNIFICANCE: In the absence of a crystal structure for any

  8. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

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    Cecilia Lässer

    2016-12-01

    Full Text Available The International Society for Extracellular Vesicles (ISEV has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs. This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge.

  9. Extracellular matrix in canine mammary tumors with special focus on versican, a versatile extracellular proteoglycan

    NARCIS (Netherlands)

    Erdélyi, Ildikó

    2006-01-01

    The extracellular matrix (ECM) research has become fundamental to understand cancer. This thesis focuses on the exploration of ECM composition and organization in canine mammary tumors, with a special interest in the large chondroitin-sulfate proteoglycan (PG), versican. Chapter 1 gives an overvie

  10. Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps.

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    Chu, Haiyan; Puchulu-Campanella, Estela; Galan, Jacob A; Tao, W Andy; Low, Philip S; Hoffman, Joseph F

    2012-07-31

    The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal-membrane complex. These pools of ATP are known to directly fuel both the Na(+)/K(+) and Ca(2+) pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na(+)/K(+) or Ca(2+) pump phosphoproteins (E(Na)-P and E(Ca)-P, respectively) from bulk [γ-(32)P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N(3)-ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N(3)-ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool's architecture can be photolabeled. With the aid of an antibody to N(3)-ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within β-spectrin, ankyrin, band 3, and GAPDH.

  11. ATP potentiates competitive inhibition of guanylyl cyclase A and B by the staurosporine analog, Gö6976: reciprocal regulation of ATP and GTP binding.

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    Robinson, Jerid W; Potter, Lincoln R

    2011-09-30

    Natriuretic peptides and ATP activate and Gö6976 inhibits guanylyl cyclase (GC)-A and GC-B. Here, the mechanism of inhibition was determined. Gö6976 progressively increased the Michaelis-Menten constant and decreased the Hill coefficient without reducing the maximal velocity of GC-A and GC-B. In the presence of 1 mm ATP, the K(i) was 1 μm for both enzymes. Inhibition of GC-B was minimal in the absence of ATP, and 1 mm ATP increased the inhibition 4-fold. In a reciprocal manner, 10 μm Gö6976 increased the potency of ATP for GC-B 4-fold. In contrast to a recent study (Duda, T., Yadav, P., and Sharma, R. K. (2010) FEBS J. 277, 2550-2553), neither staurosporine nor Gö6976 activated GC-A or GC-B. This is the first study to show that Gö6976 reduces GTP binding and the first demonstration of a competitive inhibitor of a receptor guanylyl cyclase. We conclude that Gö6976 reduces GTP binding to the catalytic site of GC-A and GC-B and that ATP increases the magnitude of the inhibition.

  12. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

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    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

  13. Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels.

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    Richter, Katrin; Kiefer, Kevin P; Grzesik, Benno A; Clauss, Wolfgang G; Fronius, Martin

    2014-01-01

    Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; Plung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.

  14. Energetic and metabolic consequences of aerobic and an-aerobic ATP-production.

    NARCIS (Netherlands)

    Schreurs, V.V.A.M.; Aarts, M.J.; IJssennagger, N.; Hermans, J.; Hendriks, W.H.

    2007-01-01

    ATP, the currency of cellular energy metabolism, can be produced during aerobic and an-aerobic oxidation of metabolic substrates. The aerobic oxidation yields CO2 + H2O as metabolic end products while ATP is produced by oxidative phosphorylation in the mitochondria. Carbohydrate, protein and fat pro

  15. Evaluation of ATP bioluminescence assays for potential use in a hospital setting.

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    Aiken, Zoie A; Wilson, Michael; Pratten, Jonathan

    2011-05-01

    ATP bioluminescence is being applied in hospitals to measure surface contamination. We compared commercial luminometers for detecting the number Staphylococcus aureus associated with surfaces. The data showed that the ATP bioluminescence methods tested were not robust enough to generate quantitative data on bacterial numbers, especially at low concentrations.

  16. Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

    NARCIS (Netherlands)

    Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar; Henning, Heiko

    2016-01-01

    Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are determined in many sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiency in nu

  17. Red blood cell ATP/ADP & nitric oxide: The best vasodilators in diabetic patients

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    Bakhtiari Nuredin

    2012-08-01

    Full Text Available Abstract Background Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose levels that result from defects in insulin secretion, or action, or both. Inspired by previous report the release of ATP from RBCs, which may participate in vessel dilation by stimulating NO production in the endothelium through purinergic receptor signaling and so, the aim of this study is to clearly determined relationship between RBC ATP/ADP ratio with nitric oxide. Methods The ATP/ADP ratio of erythrocytes among four groups of normal individuals (young & middle age, athletes’ subjects and diabetic patients were compared and the relationship between ATP/ADP ratio and NO level of plasma was determined with AVOVA test and bioluminescence method. Results ATP/ADP level in four groups normal (young & middle age, athletes, diabetes] are measured and analyzed with ANOVA test that show a significant difference between groups (P-value Conclusion In this study, a positive relationship between RBC ATP/ADP ratio and NO was found. Based on the obtained result, higher RBC ATP/ADP content may control the ratio of plasma NO in different individuals, also this results show that ATP can activate endothelial cells in NO production and is a main factor in releasing of NO from endothelial cells.

  18. Energetic and metabolic consequences of aerobic and an-aerobic ATP-production.

    NARCIS (Netherlands)

    Schreurs, V.V.A.M.; Aarts, M.J.; IJssennagger, N.; Hermans, J.; Hendriks, W.H.

    2007-01-01

    ATP, the currency of cellular energy metabolism, can be produced during aerobic and an-aerobic oxidation of metabolic substrates. The aerobic oxidation yields CO2 + H2O as metabolic end products while ATP is produced by oxidative phosphorylation in the mitochondria. Carbohydrate, protein and fat

  19. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

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    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  20. Hydrolysis and Synthesis of ATP by Membrane-Bound ATPase from a Motile Streptococcus

    NARCIS (Netherlands)

    Drift, C. van der; Janssen, D.B.; Wezenbeek, P.M.G.F. van

    1978-01-01

    ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg2+ ratio of 2. Mn2+ and Ca2+ could replace Mg2+ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide but not by sodiu

  1. Skeletal muscle mitochondria of NDUFS4(-/-) mice display normal maximal pyruvate oxidation and ATP production

    NARCIS (Netherlands)

    Alam, M.T.; Manjeri, G.R.; Rodenburg, R.J.T.; Smeitink, J.A.M.; Notebaart, R.A.; Huijnen, M.A.; Willems, P.H.G.M.; Koopman, W.J.H.

    2015-01-01

    Mitochondrial ATP production is mediated by the oxidative phosphorylation (OXPHOS) system, which consists of four multi-subunit complexes (CI-CIV) and the FoF1-ATP synthase (CV). Mitochondrial disorders including Leigh Syndrome often involve CI dysfunction, the pathophysiological consequences of whi

  2. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    Science.gov (United States)

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  3. ATP content and biomass activity in sequential anaerobic/aerobic reactors

    Institute of Scientific and Technical Information of China (English)

    陈红

    2004-01-01

    Specific ATP content of volatile solids was measured to characterize the sludge activity in a sequential anaerobic/aerobic wastewater treatment system, with an upflow anaerobic sludge blanket (UASB) reactor and a three-phase aerobic fluidized bed (AFB) reactor. The wastewater COD level was 2000(3000 mg/L in simulation of real textile wastewater. The ATP content and the specific ATP contents of volatile solids at different heights of the UASB reactor and those of the suspended and immobilized biomass in the AFB reactor were measured. In the UASB reactor, the maximum value of specific ATP (0.85 mg ATP/g VS) was obtained at a hydraulic retention time (HRT) 7.14 h in the blanket solution. In the AFB reactor, the specific ATP content of suspended biomass was higher than that of immobilized biomass and increased with hydraulic retention time reaching a maximum value of 1.6 mg ATP/g VS at hydraulic retention time 4.35 h. The ATP content of anaerobes in the UASB effluent declined rapidly under aerobic conditions following a 2nd-order kinetic model.

  4. Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation

    Science.gov (United States)

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine-5'-phosphosulfate (APS) from sulfate and ATP. To better unde...

  5. 75 FR 8467 - Airworthiness Directives; BAE SYSTEMS (Operations) Limited Model ATP Airplanes

    Science.gov (United States)

    2010-02-25

    ... (Operations) Limited Model ATP Airplanes AGENCY: Federal Aviation Administration (FAA), Department of... tasks were introduced by Service Bulletin (SB) ATP-51-002 * * *. As it was determined that these... environmental inspections for the fuselage. These additional tasks were introduced by Service Bulletin (SB)...

  6. ATP: A Coherent View for School Advanced Level Studies in Biology.

    Science.gov (United States)

    Gayford, Chris

    1986-01-01

    Discusses how instruction of biological concepts as ATP cellular energetics is related to fundamental physical science understandings. Reviews areas of common misconceptions and confusions. Summarizes results of a study which investigated students' knowledge and perception of difficulty associated with the topic of energy and ATP. (ML)

  7. ATP content and biomass activity in sequential anaerobic/aerobic reactors

    Institute of Scientific and Technical Information of China (English)

    陈红

    2004-01-01

    Specific ATP content of volatile solids was measured to characterize the sludge activity in a sequential anaerobic/aerobic wastewater treatment system, with an upflow anaerobic sludge blanket (UASB) reactor and a three-phase aerobic fluidized bed (AFB) reactor. The wastewater COD level was 2000-3000 mg/L in simulation of real textile wastewater. The ATP content and the specific ATP contents of volatile solids at different heights of the UASB reactor and those of the suspended and immobilized biomass in the AFB reactor were measured. In the UASB reactor, the maximum value of specific ATP (0.85 mg ATP/g VS) was obtained at a hydraulic retention time (HRT) 7.14 h in the blanket solution. In the AFB reactor, the specific ATP content of suspended biomass was higher than that of immobilized biomass and increased with hydraulic retention time reaching a maximum value of 1.6 mg ATP/g VS at hydraulic retention time 4.35 h. The ATP content of anaerobes in the UASB effluent declined rapidly under aerobic conditions following a 2nd-order kinetic model.

  8. Phylogeny and identification of Enterococci by atpA gene sequence analysis.

    Science.gov (United States)

    Naser, S; Thompson, F L; Hoste, B; Gevers, D; Vandemeulebroecke, K; Cleenwerck, I; Thompson, C C; Vancanneyt, M; Swings, J

    2005-05-01

    The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.

  9. An Aptamer-Based Nanobiosensor for Real-Time Measurements of ATP Dynamics

    DEFF Research Database (Denmark)

    Özalp, Cengiz; Nielsen, Lise Junker; Olsen, Lars Folke

    2010-01-01

    A nanosensor based on a new DNA aptamer can show changes in intracellular concentration of ATP of 0.5 to 8 mM and thereby the kinetics of ATP-consuming reactions in real-time. The biosensor was protected against nuclease attack by being buried in a polyacrylamide nanoparticle. This strategy can...

  10. Effect of visible laser light on ATP level of anaemic red blood cell.

    Science.gov (United States)

    Suardi, Nursakinah; Sodipo, Bashiru Kayode; Mustafa, Mohd Zulkifli; Ali, Zalila

    2016-09-01

    In this work we present influence of visible laser light on ATP level and viability of anaemic red blood cell (RBC). The visible laser lights used in this work are 460nm and 532nm. The responses of ATP level in anaemic and normal RBC before and after laser irradiation at different exposure time (30, 40, 50 and 60s) were observed. Three aliquots were prepared from the ethylenediaminetetraacetic acid (EDTA) blood sample. One served as a control (untreated) and another two were irradiated with 460nm and 560nm lasers. Packed RBC was prepared to study ATP level in the RBC using CellTiter-GloLuminescent cell Viability Assay kit. The assay generates a glow type signal produced by luciferase reaction, which is proportional to the amount of ATP present in RBCs. Paired t-test were done to analyse ATP level before and after laser irradiation. The results revealed laser irradiation improve level of ATP in anaemic RBC. Effect of laser light on anaemic RBCs were significant over different exposure time for both 460nm (p=0.000) and 532nm (p=0.003). The result of ATP level is further used as marker for RBC viability. The influence of ATP level and viability were studied. Optical densities obtained from the data were used to determine cell viability of the samples. Results showed that laser irradiation increased viability of anaemic RBC compared to normal RBC.

  11. Structural, biochemical and genetic characterization of dissimilatory ATP sulfurylase from Allochromatium vinosum.

    Directory of Open Access Journals (Sweden)

    Kristian Parey

    Full Text Available ATP sulfurylase (ATPS catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 (2- with ATP to adenosine 5'-phosphosulfate (APS and pyrophosphate (PPi. In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes.

  12. Expression of ATP7B in human gastric cardiac carcinomas in comparison with distal gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Da-Long Wu; Hui-Xing Yi; Feng-Ying Sui; Xiao-Hong Jiang; Xiao-Ming Jiang; Ying-Ying Zhao

    2006-01-01

    AIM: To analyze expression of ATP7B in gastric cardiac adenocarcinomas, its clinicopathologic significance, in comparison with distal gastric adenocarcinomas.METHODS: Immunohistochemical avidin-biotin peroxidase complex method was applied to detect the expression of ATP7B in 49 cases of cardiac carcinomas,the corresponding adjacent non-neoplastic epithelium and 55 cases of distal gastric carcinomas.RESULTS: The proportion of ATP7B positive samples in gastric cardiac carcinomas (51.0%, 25 of 49) was significantly higher than that in the corresponding adjacent non-neoplastic epithelium (22.4%, 11 of 49)(P = 0.003). ATP7B expression in poorly differentiated gastric cardiac carcinomas was significantly higher than that in well/moderately differentiated gastric cardiac carcinomas (P = 0.030). ATP7B expression in gastric cardiac carcinomas was independent of age, tumor size, nodal stage and metastasis status. ATP7B protein was detected in 30.9% (17/55 cases) of distal gastric carcinomas, markedly lower than that in gastric cardiac carcinomas (P = 0.037).CONCLUSION: ATP7B protein is frequently overexpressed in gastric cardiac carcinomas, and correlated with the differentiation of cardiac carcinoma. ATP7B expression in gastric cardiac carcinomas is significantly higher than that in distal gastric carcinomas, which might partially explain the difference of chemotherapy response and prognosis between these two gastric carcinomas.

  13. Hydrolysis and Synthesis of ATP by Membrane-Bound ATPase from a Motile Streptococcus

    NARCIS (Netherlands)

    Drift, C. van der; Janssen, D.B.; Wezenbeek, P.M.G.F. van

    1978-01-01

    ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg2+ ratio of 2. Mn2+ and Ca2+ could replace Mg2+ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide but not by sodiu

  14. Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

    Directory of Open Access Journals (Sweden)

    Askew Christopher D

    2009-12-01

    Full Text Available Abstract Background It has been proposed that adenosine triphosphate (ATP released from red blood cells (RBCs may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC. Results Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P Conclusions Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

  15. Myocardial purine metabolism : aspects of myocardial ATP metabolism and pharmacological intervention

    NARCIS (Netherlands)

    E. Harmsen (Eef)

    1984-01-01

    textabstractThe heart is obliged to contract continuously. This implicates for a human with an average heart rate of 70 beats per minute in about 70 years more than 2.5 billions of contractions. For this contraction ATP is needed as the main substrate. As a consequence continuous ATP production by t

  16. CLONING, SEQUENCING, AND EXPRESSION OF BACILLUS-SUBTILIS GENES INVOLVED IN ATP-DEPENDENT NUCLEASE SYNTHESIS

    NARCIS (Netherlands)

    KOOISTRA, J; VENEMA, G

    1991-01-01

    The genes encoding